Method and means for producing high titer, safe, recombinant lentivirus vectors

ABSTRACT

Lentiviral vectors modified at the 5′ LTR or both the 5′ and 3′ LTR are useful in the production of recombinant lentivirus vectors (See the Figure). Such vectors can be produced in the absence of a functional tat gene. Multiple transformation of the host cell with the vector carrying the transgene enhances virus production. The vectors can contain inducible or conditional promoters.

This application is a national stage application based on PCTapplication number US00/11097, filed Apr. 26, 2000, which claims thebenefit of U.S. provisional application No. 60/131,671, filed Apr. 29,1999.

FIELD OF THE INVENTION

The invention relates to novel lentiviral packaging vectors, transfervectors carrying a foreign gene of interest, stable packaging celllines, stable producer cell lines and the use thereof for producingrecombinant lentivirus in mammalian cells.

BACKGROUND OF THE INVENTION

Retrovirus vectors are a common tool for gene delivery (Miller, Nature(1992) 357:455-460). The biology of retroviral proliferation enablessuch a use. Typically, wild type full length retroviral mRNA's serveboth as a template for synthesis of viral proteins and as the viralgenome. Such mRNA's encompass a region called the encapsidation signalwhich binds certain viral proteins thereby ensuring specific associationof that mRNA with the produced virions. On infection of the target cell,reverse transcription of the retroviral mRNA into double strandedproviral DNA occurs. The retroviral enzyme, integrase, then, binds toboth long terminal repeats (LTR) which flank the proviral DNA andsubsequently catalyzes the integration thereof into the genomic DNA ofthe target cell. Integrated proviral DNA serves as the template forgeneration of new full-length retroviral mRNA's.

Retroviral vectors have been tested and found to be suitable deliveryvehicles for the stable introduction of a variety of genes of interestinto the genomic DNA of a broad range of target cells, a process knownas transduction of the cells with the gene of interest. The ability ofretrovirus vectors to deliver an unrearranged, single copy gene into abroad range of, for example, rodent, primate and human somatic cellsmakes retroviral vectors well suited for transferring genes to a cell.

A primary approach in retrovirus-derived vector design relies on removalof the encapsidation signal and sequences coding the LTR's from theviral genome without affecting viral protein expression and transfer ofsuch sequences to the construct including a nucleic acid coding the geneof interest, sometimes called the transfer vector.

A useful adjunct for producing recombinant retroviral vectors arepackaging cell lines which supply in trans the proteins necessary forproducing infectious virions, but those cells are incapable of packagingendogenous viral genomic nucleic acids (Watanabe & Temin, Molec. Cell.Biol. (1983) 3:2241-2249; Mann et al., Cell (1983) 33:153-159; andEmbretson & Temin, J. Virol. (1987) 61:2675-2683). Expression in thevector producer cells of both viral core proteins, which comprise thevirion particle, and mRNA containing LTR, encapsidation sequences andthe gene of interest, results in release by the cells of particles whichphenotypically resemble parental retrovirus, but carry the gene ofinterest instead of the viral genome. Such particles will integrate thegene of interest but not the viral DNA into the genome of target cells.

A consideration in the construction of retroviral packaging cell linesis the production of high titer vector supernatants free of recombinantreplication competent retrovirus (RCR), which have been shown to produceT cell lymphomas in rodents (Cloyd et al., J. Exp. Med. (1980)151:542-552) and in primates (Donahue et al., J. Exp. Med. (1992)176:1125-1135).

In the vector producing cells, restoration of the physical associationof LTR and encapsidation sequences with the sequences coding the viralproteins may lead to the emergence of RCR capable of self amplification.Generation of recombinant viruses during vector production is highlyundesirable for several reasons. First, the recombinant mRNA may competewith the transgene mRNA for encapsidation into virions therebydecreasing the number of transgenes per vector particle made by producercells. That competition, as well as amplification of such recombinantsin producer cells, may lead to the exponential loss of vectortransduction potential.

Second, such recombinants, if undetected during vector production, maybe introduced unintentionally to the vector recipients. There, transferof the recombinant genome to the host may cause otherwise avoidabletoxicity or an immune reaction to the transduced cells. Importantly,viral recombinants may be pathogenic or may evolve into pathogens onadditional rounds of amplification and/or through additional events ofrecombination with endogenous sequences of the host cells (such asendogenous retroviral sequences).

Recombinant retrovirus could be generated at the DNA or mRNA level. DNArecombination may take place if plasmid constructs independently codingfor packaging and transfer vector functions are mixed and co-transfectedin an attempt to create transient producer cells. To decrease the chanceof recombination at the DNA level, the constructs could be introducedinto cells one after another with concurrent selection of clones aftereach construct is associated stably with the cellular genome. Somaticcells dividing mitotically generally do not undergo crossing overbetween homologous chromosomes and since each vector constructassociation is expected to be integrated randomly into the genomic DNA,the likelihood of close association and therefore the chance ofrecombination is low.

Recombination at the mRNA level may take place during reversetranscription when both packaging mRNA and transfer vector mRNA (evenwhen generated by separated expression constructs) becomeco-encapsidated into viral particles. The retroviral enzyme reversetranscriptase (RT) uses mRNA as template for DNA synthesis. Also, RT isknown to switch between or away templates. Thus, if two different mRNA'sare present within a viral particle, when combined, a single DNA unitcould be synthesized by the RT as the result of template switching.

One approach to minimize the likelihood of generating RCR in packagingcells is to divide the packaging functions into two or more genomes, forexample, one which expresses the gag and pol gene products and the otherwhich expresses the env gene product (Bosselman et al., Molec. Cell.Biol. (1987) 7:1797-1806; Markowitz et al., J. Virol. (1988)62:1120-1124; and Danos & Mulligan, Proc. Natl. Acad. Sci. (1988)85:6460-6464). That approach minimizes the ability for co-packaging andsubsequent transfer of the two or more genomes, as well as significantlydecreasing the frequency of recombination due to the presence ofmultiple retroviral genomes in the packaging cell to produce RCR.

The rationale behind the approach of splitting the packaging functionsis that multiple recombination events must occur to generate RCR. Thatapproach, however, does not decrease the chance of individualrecombination events. Therefore partial recombinants incapable ofamplification could be generated. To monitor emergence of such partialrecombinants, novel complementing detection systems must be designed.

In the event recombinants arise, mutations (Danos & Mulligan, supra) ordeletions (Boselman et al., supra; and Markowitz et al., supra) withinvector constructs can be configured such that in the event recombinantsarise, those will be rendered non-functional. In addition, deletion ofthe 3′ LTR on both packaging constructs further reduces the ability toform functional recombinants.

It was demonstrated previously for many biological systems that thefrequency of recombination between two genetic elements is directlyproportional to the extent of homologous sequences. Thus, anotherapproach is to minimize the extent of sequence homology between andamongst the vectors. Technical difficulties associated with minimizationof the homologous sequences between transfer vector and packagingconstructs can be explained by the fact that some essential geneticelements could not be removed from at least one of the constructswithout significant loss of transduction potential.

Lentiviruses are complex retroviruses which, in addition to the commonretroviral genes gag, pol and env, contain other genes with regulatoryor structural function. The higher complexity enables the lentivirus tomodulate the life cycle thereof, as in the course of latent infection.

Lentiviruses have attracted the attention of gene therapy investigatorsbecause of the ability to integrate into non-dividing cells (Lewis etal., EMBO J. (1992) 11:3053-3058; Bukrinsky et al., Nature (1993)365:666-669; Gallay et al., Proc. Natl. Acad. Sci. USA (1997)94:9825-9830; Gallay et al., Cell (1995) 80:379-388; and Lewis et al.,J. Virol. (1994) 68:510). Replication-defective vectors from the humanlentivirus human immunodeficiency virus (HIV) transduce target cellsindependent of mitosis (Naldini et al., Science (1996) 272:263-267). Thevectors proved highly efficient for in vivo gene delivery and achievedstable long-term expression of the transgene in several target tissues,such as the brain (Naldini et al., PNAS (1996) 93:11382-1138; and Blomeret al., J. Virol. (1997) 71:6641-6649), the retina (Miyoshi et al., PNAS(1997) 94:10319-10323), the liver and the muscle (Kafri et al., NatureGenetics (1997) 17:314-317).

A typical lentivirus is HIV, the etiologic agent of AIDS. In vivo, HIVcan infect terminally differentiated cells that rarely divide, such aslymphocytes and macrophages. In vitro, HIV can infect primary culturesof monocyte-derived macrophages (MDM) as well as HeLa-Cd4 or T lymphoidcells arrested in the cell cycle by treatment with aphidicolin or γirradiation.

The complexity of the lentiviral genome may be exploited to build novelbiosafety features in the design of a retroviral vector. In addition tothe structural gag, pol and env genes common to all retroviruses, HIVcontains two regulatory genes, tat and rev, essential for viralreplication, and four accessory genes, vif, vpr, vpu and nef, that arenot crucial for viral growth in vitro but are critical for in vivoreplication and pathogenesis (Luciw., in Fields et al. (ed.), “FieldsVirology”, 3rd ed., (1996) p. 1881-1975 Lippincott-Raven Publishers,Philadelphia.).

The Tat and Rev proteins regulate the levels of HIV gene expression attranscriptional and post-transcriptional levels, respectively. Due tothe weak basal transcriptional activity of the HIV LTR, expression ofthe provirus initially results in small amounts of multiply splicedtranscripts coding for the Tat, Rev and Nef proteins. Tat dramaticallyincreases HIV transcription by binding to a stem-loop structure (TAR) inthe nascent RNA thereby recruiting a cyclin-kinase complex thatstimulates transcriptional elongation by the polymerase II complex (Weiet al., Cell (1998) 92:451-462)). Once Rev reaches a thresholdconcentration, Rev promotes the cytoplasmic accumulation of unsplicedand singly-spliced viral transcripts leading to the production of thelate viral proteins.

Rev accomplishes that effect by serving as a connector between an RNAmotif (the Rev-responsive element, RRE) found in the envelope codingregion of the HIV transcript and components of the cell nuclear exportmachinery. Only in the presence of Tat and Rev are the HIV structuralgenes expressed and new viral particles produced (Luciw, supra).

In a first generation of HIV-derived vectors (Naldini et al., Science,supra), viral particles were generated by expressing the HIV-1 coreproteins, enzymes and accessory factors from heterologoustranscriptional signals and the envelope of another virus, most oftenthe G protein of the vesicular stomatitis virus (VSV.G; Burns et al.,PNAS (1993) 90:8033-8037) from a separate plasmid.

In a second version of the system, the HIV-derived packaging componentwas reduced to the gag, pol, tat and rev genes of HIV-1 (Zufferey etal., Nat. Biotech. (1997) 15:871-875).

In either case, the vector itself carried the HIV-derived cis-actingsequences necessary for transcription, encapsidation, reversetranscription and integration (Aldovini & Young., J. Virol. (1990)64:1920-1926; Berkowitz et al., Virology (1995) 212:718-723, Kaye etal., J. Virol. (1995) 69:6588-6592; Lever et al., J. Virol. (1994) 63:4085-4087; McBride et al., J. Virol. (1989) 70:2963-2973; McBride etal., J. Virol. (1997) 71:4544-4554; Naldini et al., Science (supra); andParolin et al., J. Virol. (1994) 68: 3888-3895).

Such a vector thus encompassed from the 5′ to 3′ end, the HIV 5′ LTR,the leader sequence and the 5′ splice donor site, approximately 360 basepairs of the gag gene (with the gag reading frame closed by a syntheticstop codon), 700 base pairs of the env gene containing the RRE and asplice acceptor site, an internal promoter, for example, typically theimmediate early enhancer/promoter of human cytomegalovirus (CMV) or thatof the phosphoglycerokinase gene (PGK), the transgene and the HIV 3′LTR. Vector particles are produced by co-transfection of the constructsin 293T cells (Naldini et al., Science, supra). In that design,significant levels of transcription from the vector LTR and ofaccumulation of unspliced genomic RNA occur only in the presence of Tatand Rev.

Infection of cells is dependent on the active nuclear import of HIVpreintegration complexes through the nuclear pores of the target cells.That occurs by the interaction of multiple, partly redundant, moleculardeterminants in the complex with the nuclear import machinery of thetarget cell. Identified determinants include a functional nuclearlocalization signal (NLS) in the gag matrix (MA) protein, thekaryophilic virion-associated protein, vpr, and a C-terminalphosphotyrosine residue in the gag MA protein.

SUMMARY OF THE INVENTION

Accordingly, the instant invention relates to novel disarmed lentiviralvectors, such as packaging and transfer vectors, that direct thesynthesis of both lentiviral vector transcripts which can be packagedand lentiviral proteins for rapid production of high titer recombinantlentivirus in mammalian cells. The results are infectious particles fordelivering a foreign gene of interest to a target cell. The inventionalso provides cell lines for virus production.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts various lentivirus vectors. RSV is the Rous sarcoma virusenhancer/promoter; R is the R region of the LTR; U5 is the U5 region ofthe LTR; SD is a slice donor site, such as the HIV 5′ major splice donorsite; ψ is the Psi encapsidation signal sequence; Ga is a part of thegag gene; RRE is the rev responsive element; SA is a splice acceptorsequence; and U3 is the U3 region of the LTR.

FIG. 2 depicts additional lentivirus vectors. CMV is cytomegalovirus.Otherwise, the symbols are as found in the legend to FIG. 1.

FIG. 3 is a graph depicting graded vector production with increasingamounts of transfer vector.

FIG. 4 depicts 5′ modifications of lentivector transfer constructs.Indicated type number for a particular construct is assigned inaccordance with the removal or modification of indicated elements. Forinstance: the construct name, such as RRL7, indicates that the vector isof the type-7 construct family and can have the RSV enhancer in the U3region. Gag is the gag gene; fs is frameshift; Env OFR is the envelopegene reading frame; RRE is the Rev responsive element; SA is a spliceacceptor; and RSV is the Rous sarcoma virus.

FIG. 5 depicts schematic diagrams of novel packaging constructs. Pro isprotease; Δ env is a truncated envelope gene; pol is polymerase; poly Ais a polyadenylation site; Tet O7 is the tet regulator; MA is matrix andVSV/G is vesicular stomatitis virus G protein.

FIG. 6 depicts diagrams outlining homologous sequences between packaging(pMDLg/pRRE) and indicated transfer vector constructs. Prom is promoterand Min gag is a truncated or minimized gag.

FIG. 7 depicts diagrams outlining homologous sequences between packagingconstructs pMDLg/pRRE.2 or pMDLg/pRRE.3 and a type-7 transfer vectorconstruct. MA is matrix; CA is capsid, P2 is gag cleavage product; NC isnucleocapsid; P1 is another gag clevage product; P6 is another gagclevage protein and non-HIV Enh is a non-HIV enhancer.

FIG. 8 depicts representations of FACS (Fluorescence Activated CellSorting) plots indicating high efficiency transduction ofgrowth-arrested (by aphidicolin treatment) HeLa cells with vectorparticles produced by calcium phosphate transfection of nonoverlappinglentivector constructs. The following plasmids were transfected: 10 μgof CCL7sinCMVGFPpre, 5 μg of pMDLg/pRRE, or pMDLg/pRRE.2, orpMDLg/pRRE.3 and 3 μg of pMD.G.

FIG. 9 depicts representations of RNA protection analyses of vectorparticles obtained by transient transfection of indicated plasmids.(Plasmid pCMVΔR8.2 is described in Naldini et. al. Science, supra) Mutis mutation.

FIG. 10 depicts production and titer of vector particles produces by a2^(nd) generation packaging cell line (clone 2.54) pinged by atetracycline regulatable transfer vector.

FIG. 11 depicts a representation of a Northern analysis of transducedHeLa cells using the indicated vectors. Total RNA was assayed with a GFPspecific probe.

FIG. 12 depicts representations of FACS plots indicating that noactivation of the Tet^(o)/HIV promoter takes place on HIV-1 infection.

DETAILED DESCRIPTION OF THE INVENTION

The instant invention provides a recombinant lentivirus capable ofinfecting non-dividing cells as well as methods and means for makingsame. The virus is useful for the in vivo and ex vivo transfer andexpression of nucleic acid sequences.

The lentiviral genome and the proviral DNA have the three genes found inretroviruses: gag, pol and env, which are flanked by two LTR sequences.The gag gene encodes the internal structural (matrix, capsid andnucleocapsid) proteins; the pol gene encodes the RNA-directed DNApolymerase (reverse transcriptase), a protease and an integrase; and theenv gene encodes viral envelope glycoproteins. The 5′ and 3′ LTR's serveto promote transcription and polyadenylation of the virion RNA's. TheLTR contains all other cis-acting sequences necessary for viralreplication. Lentiviruses have additional genes including vif, vpr, tat,rev, vpu, nef and vpx (in HIV-1, HIV-2 and/or SIV).

Adjacent to the 5′ LTR are sequences necessary for reverse transcriptionof the genome (the tRNA primer binding site) and for efficientencapsidation of viral RNA into particles (the Psi site). If thesequences necessary for encapsidation (or packaging of retroviral RNAinto infectious virions) are missing from the viral genome, the cisdefect prevents encapsidation of genomic RNA. However, the resultingmutant remains capable of directing the synthesis of all virionproteins.

The invention provides a method of producing a recombinant lentiviruscapable of infecting a non-dividing cell comprising transfecting asuitable host cell with two or more vectors carrying the packagingfunctions, namely gag, pol and env, as well as rev and tat. As will bedisclosed hereinbelow, vectors lacking a functional tat gene aredesirable for certain applications. Thus, for example, a first vectorcan provide a nucleic acid encoding a viral gag and a viral pol andanother vector can provide a nucleic acid encoding a viral env toproduce a packaging cell. Introducing a vector providing a heterologousgene, herein identified as a transfer vector, into that packaging cellyields a producer cell which releases infectious viral particlescarrying the foreign gene of interest.

A lentiviral vector described herein may be packaged by threenon-overlapping expression constructs, two expressing HIV proteins andthe other the envelope of a different virus. Moreover, all HIV sequencesknown to be required for encapsidation and reverse transcription (Leveret al., supra; Aldovini & Young, supra; Kaye et al., supra; McBride &Panganiban, supra; McBride et al., supra; Parolin et al., supra; andLuciw, supra) are absent from the constructs, with the exception of theportion of the gag gene that contributes to the stem-loop structure ofthe HIV-1 packaging motif (McBride et al., supra).

A second strategy to improve vector biosafety takes advantage of thecomplexity of the lentiviral genome. The minimal set of HIV-1 genesrequired to generate an efficient vector was identified and all theother HIV reading frames were eliminated from the system. As theproducts of the removed genes are important for the completion of thevirus life cycle and for pathogenesis, no recombinant can acquire thepathogenetic features of the parental virus. All four accessory genes ofHIV could be deleted from the packaging construct without compromisinggene transduction (Zufferey et al., supra).

The tat gene is crucial for HIV replication. The tat gene product is oneof the most powerful transcriptional activators known and plays apivotal role in the exceedingly high replication rates that characterizeHIV-induced disease (Haynes et al., Science (1996) 271:324-328; Ho etal., Nature (1995) 373:123-126; and Wei et al., Nature (1995) 373117-122).

The trans-acting function of Tat becomes dispensable if part of theupstream LTR in the vector construct is replaced by constitutivelyactive promoter sequences. Furthermore, the expression of rev in transallows the production of high-titer HIV-derived vector stocks from apackaging construct which contains only gag/pol. That design makes theexpression of the packaging functions conditional on complementationavailable only in producer cells. The resulting gene delivery system,which conserves only three of the nine genes of HIV-1 and relies on fourseparate transcriptional units for the production of transducingparticles, offers significant advantages in biosafety.

Tat is required in producer cells to generate vector of efficienttransducing activity. However, that requirement can be offset byinducing constitutive high-level expression of vector RNA. Due to thelow basal transcription from the HIV LTR, Tat is necessary to increasethe abundance of vector transcripts and to allow for efficientencapsidation by the vector particles. When made in the absence of Tat,vector particles have ten-fold to twenty-fold reduced transducingactivity. However, when strong constitutive promoters replace the HIVsequence in the 5′ LTR of the transfer construct, vectors made withoutTat exhibit a less than two-fold reduction in transducing activity. AsTat strongly upregulated transcription from the chimeric LTR, thetransducing activity of the output particles must reach saturation. Theabundance of vector RNA in producer cells thus appears to be arate-limiting factor for transduction until a threshold is achieved.Conceivably, an upper limit is set by the total output of particlesavailable to encapsidate vector RNA.

Successful deletion of the tat gene was unexpected in view of a reportedadditional role for Tat in reverse transcription (Harrich et al., EMBOJ. (1997) 16:1224-1235; and Huang et al., EMBO J. (1994) 13:2886-2896).But the transduction pathway of the lentiviral vector mimics only inpart the infection pathway of HIV. The vector is pseudotyped by theenvelope of an unrelated virus and only contains the core proteins ofHIV without any accessory gene product. The VSV envelope targets thevector to the endocytic pathway and it has been shown that redirectionof HIV-1 from the normal route of entry by fusion at the plasma membranesignificantly changes the biology of the infection. For example, Nef andcyclophilin A are required for the optimal infectivity of wild-typeHIV-1 but not of a (VSV.G)HIV pseudotype (Aiken J. Virol. (1997)71:5871-5877). Also, the kinetics of reverse transcription may be morecritical for the establishment of viral infection than for genetransduction, given the differences in size and sequence between thevirus and vector genome.

Also, the Rev dependence of gag-pol expression and of the accumulationof unspliced, packageable transcripts was exploited. Yu et al. [J.Virol. (1996) 70:4530-4537] previously showed that the dependence on Revcan be used to make expression of HIV genes inducible. A core packagingsystem split in two separate non-overlapping expression constructs, onefor the gag-pol reading frames optimized for Rev-dependent expressionand the other for the Rev cDNA, therefore can be employed. Such apackaging system matches the performance of predecessors in terms ofboth yield and transducing efficiency. However, it increasessignificantly the predicted biosafety of the vector.

It has been suggested that the Rev-RRE axis could be replaced by the useof constitutive RNA transport elements of other viruses, althoughperhaps at the price of decreased efficiency (Srinivasakumar et al., J.Virol. (1997) 71:5841-5848; and Corbeau et al., Gene Ther. (1998)5:99-104). Maintaining the Rev-dependence of the system allows for anadditional level of biosafety through the splitting of the HIV-derivedcomponents of the packaging system.

The vectors per se, outside of the newly constructed vectors disclosedherein, are known in the art, see Naldini et al., Science, supra; andZufferey et al. Generally the vectors are plasmid-based or virus-based,and are configured to carry the essential sequences for incorporatingforeign nucleic acid, for selection and for transfer of the nucleic acidinto a host cell. The gag, pol and env genes of the vectors of interestalso are known in the art. Thus, the relevant genes are cloned into theselected vector and then used to transform the target cell of interest.

According to the above-indicated configuration of vectors and foreigngenes, a vector can provide a nucleic acid encoding a viral envelope(env) gene. The env gene can be derived from any virus, includingretroviruses. The env preferably is an amphotropic envelope proteinwhich allows transduction of cells of human and other species.

It may be desirable to target the recombinant virus by linkage of theenvelope protein with an antibody or a particular ligand for targetingto a receptor of a particular cell type. By inserting a sequence(including a regulatory region) of interest into the viral vector, alongwith another gene which encodes the ligand for a receptor on a specifictarget cell, for example, the vector is now target-specific. Retroviralvectors can be made target-specific by inserting, for example, aglycolipid or a protein. Targeting often is accomplished by using anantigen-binding portion of an antibody or a recombinant antibody-typemolecule, such as a single chain antibody, to target the retroviralvector. Those of skill in the art will know of, or can readily ascertainwithout undue experimentation, specific methods to achieve delivery of aretroviral vector to a specific target.

Examples of retroviral-derived env genes include, but are not limitedto: Moloney murine leukemia virus (MoMuLV or MMLV), Harvey murinesarcoma virus (HaMuSV or HSV), murine mammary tumor virus (MuMTV orMMTV), gibbon ape leukemia virus (GaLV or GALV), human immunodeficiencyvirus (HIV) and Rous sarcoma virus (RSV). Other env genes such asvesicular stomatitis virus (VSV) protein G (VSV G), that of hepatitisviruses and of influenza also can be used.

The vector providing the viral env nucleic acid sequence is associatedoperably with regulatory sequences, e.g., a promoter or enhancer. Theregulatory sequence can be any eukaryotic promoter or enhancer,including, for example, the Moloney murine leukemia viruspromoter-enhancer element, the human cytomegalovirus enhancer or thevaccinia P7.5 promoter. In some cases, such as the Moloney murineleukemia virus promoter-enhancer element, the promoter-enhancer elementsare located within or adjacent to the LTR sequences.

Preferably, the regulatory sequence is one which is not endogenous tothe lentivirus from which the vector is being constructed. Thus, if thevector is being made from SIV, the SIV regulatory sequence found in theSIV LTR would be replaced by a regulatory element which does notoriginate from SIV.

While VSV G protein is a desirable env gene because VSV G confers broadhost range on the recombinant virus, VSV G can be deleterious to thehost cell. Thus, when a gene such as that for VSV G is used, it ispreferred to employ an inducible promoter system so that VSV Gexpression can be regulated to minimize host toxicity when VSV G isexpression is not required.

For example, the tetracycline-regulatable gene expression system ofGossen & Bujard (Proc. Natl. Acad. Sci. (1992) 89:5547-5551) can beemployed to provide for conditional or inducible expression of VSV Gwhen tetracycline is withdrawn from the transferred cell. Thus, thetet/VP16 transactivator is present on a first vector and the VSV Gcoding sequence is cloned downstream from a promoter controlled by tetoperator sequences on another vector.

Such a hybrid promoter can be inserted in place of the 3′ U3 region ofthe LTR of a transfer vector. As a result of transduction of targetcells by the vector particles produced by the use of such a transfervector, the hybrid promoter will be copied to the 5′ U3 region onreverse transcription. In the target cells, such a conditionalexpression of a gene can be activated to express full-length packagablevector transcripts only in the presence of tTA—for example, aftertransduction of an appropriate packaging cell line expressing tTA.

Use of such vectors in producer cells allows one to “turn on” theproduction of the packagable vector mRNA messages at high levels onlywhen needed. In contrast, on transduction of cells which do not expresstTA, the hybrid promoter becomes transcriptionally silent. Suchtranscriptional silence was maintained even in the presence of HIV Tatprotein, which is known to be capable of upregulating basaltranscriptional activity of heterologous promoters. The promoter systemsignificantly reduces the chance of mobilization of the vector genomeeven if transduced cells are infected by wild type HIV-1.

Another embodiment relates to a retroviral vector system based onlentivirus in which sequence homology (sequence overlap) between codingsequences of packaging and transfer vector constructs is eliminated.Importantly, vector particles produced by the use of such constructsretain high levels of transduction potential. Use of such constructs ina vector production system is expected to most significantly decreasethe frequency of recombination events, which is a significant advance inbiosafety associated with such a vector system.

It is known that throughout the gag-pol coding mRNA, several cis-actingrepression sequences (CRS) are present. The sequences prevent transportof mRNA's to the cell cytoplasm and therefore prevent encoded proteinexpression. To suppress the action of CRS, HIV-1 mRNA's contain ananti-repression signal called RRE to which Rev protein may bind. HIV-1mRNA-Rev complexes then are efficiently transported to the cellcytoplasm where the complex dissociates and mRNA becomes available fortranslation.

At least two approaches are available for choosing the minimal amountsof HIV sequences necessary in Gag and Gag-Pol expressing packagingvectors. First, only the gag-pol gene could be inserted. In that case,all, or at least most of the CRS will need to be identified and mutatedwithout effecting the encoded amino acid sequence. If that isaccomplished, the Rev gene can be eliminated from the vector system.

Second, the minimal RRE element can be introduced to the gag-polexpression cassette so that the sequence thereof will be part of theresulting mRNA. In that case, expression of Gag and Gag-Pol polyproteinswill require presence of the anti-repressor, Rev. Rev protein itself,however, does not need to be part of the gag-pol expression vector butcould be provided in trans from independent and, preferably,nonoverlapping with the gag-pol expression cassette.

In the system where Rev protein is not required for efficient productionof transfer vector mRNA, the rev gene and RRE element may be eliminatedfrom the vector system as a further biosafety measure. In such a system,however, if the gag-pol gene in whole or in part is transferred into avector recipient as the result of a homologous or a non-homologousrecombination event, the expression may occur.

In contrast, a vector system in which gag-pol gene expression isdependent on Rev may be a valuable safety alternative. Thus, if a Revutilizing vector system is designed so all of the components do not havehomologous sequences, in the unlikely event of recombination, whichwould result in transfer the of gag-pol sequences to the vectorrecipient, the expression thereof is much less likely to occur since thetransferred recombinant must contain both the RRE element as well as Revcoding sequence capable of being expressed.

Suitable vectors are the type-7 vectors which in comparison to type-2vectors, integrate further modification of HIV-1 sequences: one basemutation within the SD site to prevent splicing of full length mRNA;absence of the HIV-1 SA site and flanking sequences; contains only 43bases of 5′ gag ORF; and absence of the RRE element. The type-7 vectorsencompass only 43 bases homologous to pMDLg/pRRE and no homology topMDLg/pRRE.2 and pMDLg/pRRE.3 packaging vectors. Table 1 below providesan example of vector titer yields obtained by transfection of thedescribed minimally overlapping and nonoverlapping constructs.

TABLE 1 Rev expressing VSV/G expressing Titer Packaging Vector Transfervector Plasmid plasmid (Transducing Units (12 μg of plasmid DNA (10 μgof plasmid DNA (2.5 μg of plasmid (3.5 μg of plasmid per 1 ml oftransfected) transfected) DNA transfected) DNA transfected) supernatant)pMDLg/pRRE pCCL7sinCMVGFPpre PRSV-Rev pMD.G 4.71 × 10⁶ pMDLg/pRRE.2pCCL7sinCMVGFPpre pRSV-Rev pMD.G 2.74 × 10⁶ pMDLg/pRRE.3pCCL7sinCMVGFPpre pRSV-Rev pMD.G 1.16 × 10⁶

As the main property of interest for HIV-derived vectors is the abilityto transduce nondividing and slowly dividing cells and tissues,nonoverlapping vectors were tested for transduction in cell cyclearrested cells. In contrast to MoMLV vectors, minimal HIV-derivedvectors maintained transduction potential in both dividing and growtharrested cells.

Furthermore, an HIV-1 RNA element present in the packaging vectorgag-pol mRNA was observed to lead to specific encapsidation ofsignificant amounts of the message into released vector particles undercertain conditions. The element serves as the HIV-1 major splice donorsite (SD) and consists of at least nucleotides, GACUGGUGAG (SEQ ID NO:1). In the absence of transfer vector expression, vector particlesgenerated only by pMDLg/pRRE packaging construct have no detectablegag-pol RNA message. Analysis of total RNA extracted from the cellswhich produced the vector particles, showed that expression levels inall cases were similar. When 5′mRNA regions of the tested packagingvectors were compared, it became apparent that the specified abovesequence is the determinant which provides specific encapsidation of themessages.

The heterologous or foreign nucleic acid sequence, the transgene, islinked operably to a regulatory nucleic acid sequence As used herein,the term “heterologous” nucleic acid sequence refers to a sequence thatoriginates from a foreign species, or, if from the same species, it maybe substantially modified from the original form. Alternatively, anunchanged nucleic acid sequence that is not expressed normally in a cellis a heterologous nucleic acid sequence.

The term “operably linked” refers to functional linkage between aregulatory sequence and a heterologous nucleic acid sequence resultingin expression of the latter. Preferably, the heterologous sequence islinked to a promoter, resulting in a chimeric gene. The heterologousnucleic acid sequence is preferably under control of either the viralLTR promoter-enhancer signals or of an internal promoter, and retainedsignals within the retroviral LTR can still bring about efficientexpression of the transgene.

The foreign gene can be any nucleic acid of interest which can betranscribed. Generally the foreign gene encodes a polypeptide.Preferably the polypeptide has some therapeutic benefit. The polypeptidemay supplement deficient or nonexistent expression of an endogenousprotein in a host cell. The polypeptide can confer new properties on thehost cell, such as a chimeric signalling receptor, see U.S. Pat. No.5,359,046. The artisan can determine the appropriateness of a foreigngene practicing techniques taught herein and known in the art. Forexample, the artisan would know whether a foreign gene is of a suitablesize for encapsidation and whether the foreign gene product is expressedproperly.

It may be desirable to modulate the expression of a gene regulatingmolecule in a cell by the introduction of a molecule by the method ofthe invention. The term “modulate” envisions the suppression ofexpression of a gene when it is over-expressed or augmentation ofexpression when it is under-expressed. Where a cell proliferativedisorder is associated with the expression of a gene, nucleic acidsequences that interfere with the expression of a gene at thetranslational level can be used. The approach can utilize, for example,antisense nucleic acid, ribozymes or triplex agents to blocktranscription or translation of a specific mRNA, either by masking thatmRNA with an antisense nucleic acid or triplex agent, or by cleavingsame with a ribozyme.

Antisense nucleic acids are DNA or RNA molecules which are complementaryto at least a portion of a specific mRNA molecule (Weintraub, Sci. Am.(1990) 262:40). In the cell, the antisense nucleic acids hybridize tothe corresponding mRNA forming a double-stranded molecule. The antisensenucleic acids interfere with the translation of the mRNA since the cellwill not translate a mRNA that is double-stranded. Antisense oligomersof about 15 nucleotides or more are preferred since such are synthesizedeasily and are less likely to cause problems than larger molecules whenintroduced into the target cell. The use of antisense methods to inhibitthe in vitro translation of genes is well known in the art(Marcus-Sakura, Anal. Biochem. (1988) 172:289).

The antisense nucleic acid can be used to block expression of a mutantprotein or a dominantly active gene product, such as amyloid precursorprotein that accumulates in Alzheimer's disease. Such methods are alsouseful for the treatment of Huntington's disease, hereditaryParkinsonism and other diseases. Antisense nucleic acids are also usefulfor the inhibition of expression of proteins associated with toxicity.

Use of an oligonucleotide to stall transcription can be by the mechanismknown as the triplex strategy since the oligomer winds arounddouble-helical DNA, forming a three-strand helix. Therefore, the triplexcompounds can be designed to recognize a unique site on a chosen gene(Maher et al., Antisense Res and Dev. (1991) 1(3):227; Helene,Anticancer Drug Dis. (1991) 6(6):569).

Ribozymes are RNA molecules possessing the ability to specificallycleave other single-stranded RNA in a manner analogous to DNArestriction endonucleases. Through the modification of nucleotidesequences which encode those RNA's, it is possible to engineer moleculesthat recognize and cleave specific nucleotide sequences in an RNAmolecule (Cech, J. Amer. Med Assn. (1988) 260:3030). A major advantageof that approach is only mRNA's with particular sequences areinactivated.

It may be desirable to transfer a nucleic acid encoding a biologicalresponse modifier. Included in that category are immunopotentiatingagents including nucleic acids encoding a number of the cytokinesclassified as “interleukins”, for example, interleukins 1 through 12.Also included in that category, although not necessarily workingaccording to the same mechanism, are interferons, and in particulargamma interferon (γ-IFN), tumor necrosis factor (TNF) andgranulocyte-macrophage-colony stimulating factor (GM-CSF). It may bedesirable to deliver such nucleic acids to bone marrow cells ormacrophages to treat inborn enzymatic deficiencies or immune defects.Nucleic acids encoding growth factors, toxic peptides, ligands,receptors or other physiologically important proteins also can beintroduced into specific non-dividing cells.

Thus, the recombinant lentivirus of the invention can be used to treatan HIV-infected cell (e.g., T-cell or macrophage) with an anti-HIVmolecule. In addition, respiratory epithelium, for example, can beinfected with a recombinant lentivirus of the invention having a genefor cystic fibrosis transmembrane conductance regulator (CFTR) fortreatment of cystic fibrosis.

The method of the invention may also be useful for neuronal, glial,fibroblast or mesenchymal cell transplantation, or “grafting”, whichinvolves transplantation of cells infected with the recombinantlentivirus of the invention ex vivo, or infection in vivo into thecentral nervous system or into the ventricular cavities or subdurallyonto the surface of a host brain. Such methods for grafting will beknown to those skilled in the art and are described in Neural Graftingin the Mammalian CNS, Bjorklund & Stenevi, eds. (1985).

For diseases due to deficiency of a protein product, gene transfer couldintroduce a normal gene into the affected tissues for replacementtherapy, as well as to create animal models for the disease usingantisense mutations. For example, it may be desirable to insert a FactorVIII or IX encoding nucleic acid into a lentivirus for infection of amuscle, spleen or liver cell.

The promoter sequence may be homologous or heterologous to the desiredgene sequence. A wide range of promoters may be utilized, including aviral of a mammalian promoter. Cell or tissue specific promoters can beutilized to target expression of gene sequences in specific cellpopulations. Suitable mammalian and viral promoters for the instantinvention are available in the art. A suitable promoter is one which isinducible or conditional.

Optionally during the cloning stage, the nucleic acid construct referredto as the transfer vector, having the packaging signal and theheterologous cloning site, also contains a selectable marker gene.Marker genes are utilized to assay for the presence of the vector, andthus, to confirm infection and integration. The presence of a markergene ensures the selection and growth of only those host cells whichexpress the inserts. Typical selection genes encode proteins that conferresistance to antibiotics and other toxic substances, e.g., histidinol,puromycin, hygromycin, neomycin, methotrexate etc. and cell surfacemarkers.

The recombinant virus of the invention is capable of transferring anucleic acid sequence into a mammalian cell. The term, “nucleic acidsequence”, refers to any nucleic acid molecule, preferably DNA, asdiscussed in detail herein. The nucleic acid molecule may be derivedfrom a variety of sources, including DNA, cDNA, synthetic DNA, RNA orcombinations thereof. Such nucleic acid sequences may comprise genomicDNA which may or may not include naturally occurring introns. Moreover,such genomic DNA may be obtained in association with promoter regions,poly A sequences or other associated sequences. Genomic DNA may beextracted and purified from suitable cells by means well known in theart. Alternatively, messenger RNA (mRNA) can be isolated from cells andused to produce cDNA by reverse transcription or other means.

Preferably, the recombinant lentivirus produced by the method of theinvention is a derivative of human immunodeficiency virus (HIV). The envwill be derived from a virus other than HIV.

The method of the invention provides, in some embodiments, three vectorswhich provide all of the functions required for packaging of recombinantvirions, such as, gag, pol, env, tat and rev, as discussed above. Asnoted herein, tat may be deleted functionally for unexpected benefits.There is no limitation on the number of vectors which are utilized solong as the vectors are used to transform and to produce the packagingcell line to yield recombinant lentivirus.

The vectors are introduced via transfection or infection into thepackaging cell line. The packaging cell line produces viral particlesthat contain the vector genome. Methods for transfection or infectionare well known by those of skill in the art. After co-transfection ofthe packaging vectors and the transfer vector to the packaging cellline, the recombinant virus is recovered from the culture media andtitered by standard methods used by those of skill in the art.

Thus, the packaging constructs can be introduced into human cell linesby calcium phosphate transfection, lipofection, electroporation or othermethod, generally together with a dominant selectable marker, such asneo, DHFR, Gln synthetase or ADA, followed by selection in the presenceof the appropriate drug and isolation of clones. The selectable markergene can be linked physically to the packaging genes in the construct.

Stable cell lines wherein the packaging functions are configured to beexpressed by a suitable packaging cell are known. For example, see U.S.Pat. No. 5,686,279; and Ory et al., Proc. Natl. Acad. Sci. (1996)93:11400-11406, which describe packaging cells.

Zufferey et al., supra, teach a lentiviral packaging plasmid whereinsequences 3′ of pol including the HIV-1 env gene are deleted. Theconstruct contains tat and rev sequences and the 3′ LTR is replaced withpoly A sequences. The 5′ LTR and psi sequences are replaced by anotherpromoter, such as one which is inducible. For example, a CMV promoter orderivative thereof can be used.

The packaging vectors of interest contain additional changes to thepackaging functions to enhance lentiviral protein expression and toenhance safety. For example, all of the HIV sequences upstream of gagcan be removed. Also, sequences downstream of env can be removed.Moreover, steps can be taken to modify the vector to enhance thesplicing and translation of the RNA.

To provide a vector with an even more remote possibility of generatingreplication competent lentivirus, the instant invention provides forlentivirus packaging plasmids wherein tat sequences, a regulatingprotein which promotes viral expression through a transcriptionalmechanism, are deleted functionally. Thus, the tat gene can be deleted,in part or in whole, or various point mutations or other mutations canbe made to the tat sequence to render the gene non-functional. Anartisan can practice known techniques to render the tat genenon-functional.

The techniques used to construct vectors, and to transfect and to infectcells, are practiced widely in the art. Practitioners are familiar withthe standard resource materials which describe specific conditions andprocedures. However, for convenience, the following paragraphs may serveas a guideline.

Construction of the vectors of the invention employs standard ligationand restriction techniques which are well understood in the art (seeManiatis et al., in Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory, N.Y., 1982). Isolated plasmids, DNA sequences orsynthesized oligonucleotides are cleaved, tailored and religated in theform desired.

Site-specific DNA cleavage is performed by treating with the suitablerestriction enzyme (or enzymes) under conditions which are understood inthe art, and the particulars of which are specified by the manufacturerof the commercially available restriction enzymes, see, e.g. New EnglandBiolabs, Product Catalog. In general, about 1 μg of plasmid or DNAsequences is cleaved by one unit of enzyme in about 20 μl of buffersolution. Typically, an excess of restriction enzyme is used to ensurecomplete digestion of the DNA substrate. Incubation times of about onehour to two hours at about 37° C. are workable, although variations canbe tolerated. After each incubation, protein is removed by extractionwith phenol/chloroform, which may be followed by ether extraction, andthe nucleic acid recovered from aqueous fractions by precipitation withethanol. If desired, size separation of the cleaved fragments may beperformed by polyacrylamide gel or agarose gel electrophoresis usingstandard techniques. A general description of size separations is foundin Methods of Enzymology 65:499-560 (1980).

Restriction cleaved fragments may be blunt ended by treating with thelarge fragment of E. coli DNA polymerase I (Klenow) in the presence ofthe four deoxynucleotide triphosphates (dNTP's) using incubation timesof about 15 to 25 minutes at 20° C. in 50 mM Tris (pH 7.6) 50 mM NaCl, 6mM MgCl2, 6 mM DTT and 5-10 μM dNTP's. The Klenow fragment fills in at5′ sticky ends but chews back protruding 3′ single strands, even thoughthe four dNTP's are present. If desired, selective repair can beperformed by supplying only one of the dNTP's, or with selected dNTP's,within the limitations dictated by the nature of the sticky ends. Aftertreatment with Klenow, the mixture is extracted with phenol/chloroformand ethanol precipitated. Treatment under appropriate conditions with S1nuclease or Bal-31 results in hydrolysis of any single-stranded portion.

Ligations can be performed in 15-50 μl volumes under the followingstandard conditions and temperatures: 20 mM Tris-Cl pH 7.5, 10 mM MgCl2,10 mM DTT, 33 mg/ml BSA, 10 mM-50 mM NaCl and either 40 μM ATP,0.01-0.02 (Weiss) units T4 DNA ligase at 0° C. (for “sticky end”ligation) or 1 mM ATP, 0.3-0.6 (Weiss) units T4 DNA ligase at 14° C.(for “blunt end” ligation). Intermolecular “sticky end” ligations areusually performed at 33-100 μg/ml total DNA concentrations (5-100 mMtotal end concentration). Intermolecular blunt end ligations (usuallyemploying a 10-30 fold molar excess of linkers) are performed at 1 μMtotal ends concentration.

Thus, according to the instant invention, a lentiviral packaging vectoris made to contain a promoter and other optional or requisite regulatorysequences as determined by the artisan, gag, pol, rev, env or acombination thereof, and with specific functional or actual excision oftat, and optionally other lentiviral accessory genes.

Lentiviral transfer vectors (Naldini et al., Science supra; Proc. Natl.Acad. Sci., supra) have been used to infect human cells growth-arrestedin vitro and to transduce neurons after direct injection into the brainof adult rats. The vector was efficient at transferring marker genes invivo into the neurons and long term expression in the absence ofdetectable pathology was achieved. Animals analyzed ten months after asingle injection of the vector, the longest time tested so far, showedno decrease in the average level of transgene expression and no sign oftissue pathology or immune reaction. (Blomer et al., supra). An improvedversion of the lentiviral vector in which the HIV virulence genes env,vif, vpr, vpu and nef were deleted without compromising the ability ofthe vector to transduce non-dividing cells have been developed. Themultiply attenuated version represents a substantial improvement in thebiosafety of the vector (Zufferey et al., supra).

In transduced cells, the integrated lentiviral vector generally has anLTR at each termini. The 5′ LTR may cause accumulation of “viral”transcripts that may be the substrate of recombination, in particular inHIV-infected cells. The 3′ LTR may promote downstream transcription withthe consequent risk of activating a cellular protooncogene.

The U3 sequences comprise the majority of the HIV LTR. The U3 regioncontains the enhancer and promoter elements that modulate basal andinduced expression of the HIV genome in infected cells and in responseto cell activation. Several of the promoter elements are essential forviral replication. Some of the enhancer elements are highly conservedamong viral isolates and have been implicated as critical virulencefactors in viral pathogenesis. The enhancer elements may act toinfluence replication rates in the different cellular target of thevirus (Marthas et al., J. Virol. (1993) 67:6047-6055).

As viral transcription starts at the 3′ end of the U3 region of the 5′LTR, those sequences are not part of the viral mRNA and a copy thereoffrom the 3′ LTR acts as template for the generation of both LTR's in theintegrated provirus. If the 3′ copy of the U3 region is altered in aretroviral vector construct, the vector RNA still is produced from theintact 5′ LTR in producer cells, but cannot be regenerated in targetcells. Transduction of such a vector results in the inactivation of bothLTR's in the progeny virus. Thus, the retrovirus is self-inactivating(SIN) and those vectors are known as Sin transfer vectors.

There are, however, limits to the extent of the deletion at the 3′ LTR.First, the 5′ end of the U3 region serves another essential function invector transfer, being required for integration (terminaldinucleotide+att sequence). Thus, the terminal dinucleotide and the attsequence may represent the 5′ boundary of the U3 sequences which can bedeleted. In addition, some loosely defined regions may influence theactivity of the downstream polyadenylation site in the R region.Excessive deletion of U3 sequence from the 3′ LTR may decreasepolyadenylation of vector transcripts with adverse consequences both onthe titer of the vector in producer cells and the transgene expressionin target cells. On the other hand, limited deletions may not abrogatethe transcriptional activity of the LTR in transduced cells.

New versions of a lentivirus transfer vector described herein carryincreasing deletions of the U3 region of the 3′ LTR (FIG. 1: the U3deletions span from nucleotide-418 of the U3 LTR to the indicatedposition: SIN-78, SIN-45, SIN-36 and SIN-18). Lentiviral vectors withalmost complete deletion of the U3 sequences from the 3′ LTR weredeveloped without compromising either the titer of vector in producercells or transgene expression in target cells. The most extensivedeletion (−418 to −18) extends as far as to the TATA box, thereforeabrogating any transcriptional activity of the LTR in transduced cells.Thus, the lower limit of the 3′ deletion may extend as far as includingthe TATA box. The deletion may be of the remainder of the U3 region upto the R region. That represents a dramatic gain in vector safety. Thevarious deletions were produced practicing methods known in the art.

Surprisingly, the average expression level of the transgene was evenhigher in cells transduced by the SIN vectors as compared to more intactvectors. That was probably due to the removal of transcriptionalinterference from the upstream HIV LTR on the internal promoter.SIN-type vectors with such extensive deletions of the U3 region couldnot be generated for murine leukemia virus (MLV)-based retroviralvectors without compromising efficiency of transduction.

The 5′ LTR of transfer vector construct was modified by substitutingpart or all of the transcriptional regulatory elements of the U3 regionwith heterologous enhancer/promoters. The changes were made to enhancethe expression of transfer vector RNA in producer cells; to allow vectorproduction in the absence of the HIV tat gene; and to remove theupstream wild-type copy of the HIV LTR that can recombine with the 3′deleted version to “rescue” the above described SIN vectors.

Thus, vectors containing the above-described alterations at the 5′ LTR,5′ vectors, can find use as transfer vectors because of the sequences toenhance expression and in combination with packaging cells that do notexpress tat.

Such 5′ vectors can also carry modifications at the 3′ LTR as discussedhereinabove to yield improved transfer vectors which have not onlyenhanced expression and can be used in packaging cells that do notexpress tat but can be self-inactivating as well.

The transcription from the HIV LTR is highly dependent on thetransactivator function of the tat protein. In the presence of tat,often expressed by the core packaging construct existing in producercells, vector transcription from the HIV LTR is stimulated strongly. Asthat full-length “viral” RNA has a full complement of packaging signals,the RNA is encapsidated efficiently into vector particles andtransferred to target cells. The amount of vector RNA available forpackaging in producer cells is a rate-limiting step in the production ofinfectious vector.

The enhancer or the enhancer and promoter regions of the 5′ LTR weresubstituted with the enhancer or the enhancer and promoter of the humancytomegalovirus (CMV) or Rous sarcoma virus (RSV), respectively, seeFIG. 2 for a schematic of the constructs and the code names of thehybrid vectors. The CCL and RRL vectors have complete substitution ofthe 5′ U3 region.

The control lentivector HR2 and the panel of 5′ hybrids were compared inproducer cells transfected with the transfer vector, and with or withoutpackaging constructs, which provide the tat transactivator. Thetranscriptional level of the four chimeric vectors is higher than thatof a control lentivector both in the presence and in the absence of thepackaging construct. All chimeric vectors efficiently transfer thetransgene into target cells and the RRL vector performs as well as thecontrol HR2 vector. Finally, integration of the vector in target cellswas confirmed by examining transduced cells at an early and a laterpassage after transduction. No decrease was observed in the percentageof transgene-positive cells indicating that the vector had beenintegrated.

The high level of expression of the 5′ LTR modified transfer vector RNAobtained in producer cells in the absence of a packaging constructindicates the producing vector is functional in the absence of afunctional tat gene. Functional deletion of the tat gene as indicatedfor the packaging plasmid disclosed hereinabove would confer a higherlevel of biosafety to the lentiviral vector system given the number ofpathogenetic activities associated with the tat protein. Thus, alentiviral vector of significantly improved biosafety is a SIN transfervector that has no wild-type copy of the HIV LTR either at the 5′ or atthe 3′ end, which is used in conjunction with tat-less packaging vectorsas described herein.

Viral supernatants are harvested using standard techniques such asfiltration of supernatants 48 hours post transfection. The viral titeris determined by infection of, for example, 106 NIH 3T3 cells or 105HeLa cells with an appropriate amount of viral supernatant, in thepresence of 8 g/ml polybrene (Sigma Chemical Co., St. Louis, Mo.).Forty-eight hours later, the transduction efficiency is assayed.

Thus, the instant invention provides methods and means for producinghigh titer recombinant virus. Those virus particle preparations can beused to infect target cells using techniques known in the art. Thus theinstant invention will find use in ex vivo gene therapy applicationswherein target cells are removed from a host, transformed in culturepracticing known techniques and then returned to the host.

The invention now having been described in detail, provided hereinbeloware non-limiting examples demonstrating various embodiments of theinstant invention.

EXAMPLE 1 Construction of Lentiviral Packaging Plasmids

The lentiviral packaging plasmids were derived from the plasmidpCMVΔR8.9 (ΔVprΔVifΔVpuΔNef) described previously in Zufferey et al.,supra. All the remaining sequences of the nef gene in pCMVΔR8.9 wereremoved by digesting with XhoI and BstEII, filling in with Klenow andreligating. The construction deleted 100 basepairs, joining thetruncated env reading frame of HIV-1 to the genomic insulinpolyadenylation site and yielding the plasmid pCMVΔR8.73.

In another embodiment of the invention, 133 basepairs of CMV-derivedsequences downstream of the CMV promoter were deleted in the plasmidpCMVΔR8.73. That sequence contains a splice donor site and it wasremoved by digestion of the plasmid pCMVΔR8.73 with SacII and religationof the larger fragment, obtaining the plasmid pCMVΔR8.74.

In another embodiment of the invention, all the HIV-derived sequencesremaining in the plasmid pCMVΔR8.74 upstream of the initiating codon ofthe gag gene were removed, except for the consensus 5′splice donor site.At the same time, the sequence upstream of the gag gene was changed foroptimal translation efficiency obtaining the plasmid pCMVΔR8.75.pCMVΔR8.75 was derived from pCMVΔR8.74 by replacing the 94 bp SstII-ClaIfragment with an SstII-ClaI oligonucleotide linker consisting of,5′-GGGACTGGTGAGTGAATTCGAGATCTGCCGCCGCCATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGAT-3′ (SEQ ID NO: 2) and5′-CGATCTAATTCTCCCCCGCTTAATACTGACGCTCTCGCACCCATGGCGGCGGCAGATCTCGAATFCACTCACCAGTCCCGC-3′ (SEQ ID NO: 3).

In another embodiment of the invention, an inducible packaging constructwas obtained by replacing the PstI-SacII fragment of pCMVΔR8.74containing the CMV promoter with seven tandem copies of the tetracyclineoperator sequences linked to a minimal CMV promoter. The tet-regulatedpackaging plasmid pTetΔR8.74 was obtained.

EXAMPLE 2 Construction of Lentiviral Transfer Vectors

The lentiviral transfer vector plasmids were derived from the plasmidpHR′-CMV-LacZ described previously in Naldini et al. Science, supra.pHR2 is a lentiviral transfer vector in which 124 bp of nef sequencesupstream of the 3′LTR in pHR′ were replaced with a polylinker both toreduce HIV1 sequences and to facilitate transgene cloning. pHR2 wasderived from pHR′-CMV-LacZ by replacing the 4.6 kb ClaI-Stul fragmentwith the 828 bp ClaI-StuI fragment generated by PCR using pHR′-CMV-LacZas the template and the oligonucleotide,5′-CCATCGATCACGAGACTAGTCCTACGTATCCCCGGGGACGGGATCCGCGGA ATTCCGTTTAAGAC-3′(SEQ ID NO: 4) and 5′-TTATAATGTCAAGGCCTCTC-3′ (SEQ ID NO: 5) in athree-part ligation with a 4.4 kb StuI-NcoI fragment and a 4.5 kbNcoI-ClaI fragment from pHR′-CMV-LacZ.

In another embodiment of the invention, pHR3 is a lentiviral transfervector in which 148 bp of env coding sequences (including an ATG)upstream of the Rev Response Element (RRE) in pHR2 were deleted. pHR3was derived from pHR2 by replacing the 893 bp NotI-SpeI fragment of pHR2with a 747 bp NotI-SpeI fragment generated by PCR using pHR2 as thetemplate with oligonucleotide primers 5′-GCGGCCGCAGGAGCTTTGTTCCTTGG-3′(SEQ ID NO: 6) and 5′-TACGTAGGACTAGTCTCG-3′ (SEQ ID NO: 7).

In another embodiment of the invention, pHR5 is a lentiviral transfervector in which 310 bp gag coding sequences (all gag coding sequencesdownstream from amino acid 15 of the Gag protein) were deleted frompHR2. pHR5 was derived by digestion of pHR2 with NruI, addition of aNotI linker (synthetic oligonucleotide 5′-TTGCGGCCGCAA-3′; SEQ ID NO:8), digestion with NotI to excise the 310 bp fragment, followed byreligation.

In another embodiment of the invention, pHR6 is a lentiviral vector inwhich 5′ splice donor signal was mutated (TGGT to TGAT) to enhanceproduction of full-length transcripts capable of being packaged. pHR6was derived from pHR5 by replacing the 239 bp AflII-ApoI fragment with a239 bp AflII-ApoI fragment generated by PCR using a pHR2 as the templatewith oligonucleotide primers 5′-CCACTGCTFAAGCCT-3′ (SEQ ID NO:9) andS′CAAAATTZ1′TGGCGTACTCATCAGTCGCCGCCCCTCG-3′ (SEQ ID NO:10).

All PCR fragments were generated by first cloning the PCR reactionproduct directly into the TA cloning vector pCR2.1 (Invitrogen) followedby sequence verification and excision with the appropriate enzymes.

EXAMPLE 3 Construction of 5′ LTR Chimeric Lentiviral Transfer Vectors

In another embodiment of the invention, the 5′ LTR of the lentiviralvector contains the enhancer and promoter from the U3 region of the RousSarcoma Virus (RSV) joined to the R region of HIV-1 (plasmid pRRL).

pRRL is a lentiviral transfer vector in which the enhancer and promoter(nucleotides −233 to −1 relative to the transcriptional start site) ofRSV is precisely fused to the R region of HIV-1 using an oligonucleotidelinker. pRRL was derived from plasmids pRT43.RSV.F3, see WO97/07225, andpHR2 by replacing the 3.4 kb EcoRI-HpaI fragment of pRT43.RSV.F3 withthe 0.67 kb BglII-NotI fragment from pHR2 and the 1.7 kb NotI-StuIfragment from pHR2 along with a synthetic EcoRI-BglII oligonucleotidelinker consisting of oligonucleotides5′-AATTGCCGCATTGCAGAGATATTGTATTTAAGTGCCTAGCTCGATACAATAAACGGGTCTCTCTGGTTAGACCA-3′ (SEQ ID NO:11) and5′-GATCTGGTCTAACCAGAGAGACCCGTTTATTGTATCGAGCTAGGCACTTAAATACAATATCTCTGCAATGCGGC-3′ (SEQ ID NO:12).

In another embodiment of the invention, the 5′ LTR of the lentiviralvector contains the enhancer (nucleotides −233-−50 relative to thetranscriptional start site) of the Rous Sarcoma Virus (RSV) joined tothe promoter region (from the position −78 bp relative to thetranscriptional start site) of HIV-1 (plasmid pRLL).

pRLL is a lentiviral transfer vector in which the enhancer of RSV isfused to the promoter region of HIV-1 using an oligonucleotide linker.pRRL was derived from plasmids pRT43.RSV.F3 and pHR2 by replacing the3.4 kb EcoRI-HpaI fragment of pRT43.RSV.F3 with the 0.724 kb AlwNI-NotIfragment from pHR2 and the 1.7 kb NotI-StuI fragment from pHR2 alongwith a synthetic EcoRI-AlwNI oligonucleotide linker consisting of theoligo, 5′-AATFGGAGGCGTGGCCTGGGCGGGACTGGGGAGTGGCGAGCCCTCAGATC-3′ (SEQ IDNO:13) and the oligonucleotide,5′-CTGAGGGCTCGCCACTCCCCAGTCCCGCCCAGGCCACGCCTCC-3′ (SEQ ID NO:14).

In another embodiment of the invention (plasmid pCCL), the 5′ LTR of thelentiviral vector contains the immediate early enhancer and promoter(nucleotides −673 to −1, relative to the transcriptional start siteaccording to Boshart et al. (Cell (1985) 41: 521-530), of humancytomegalovirus (CMV) joined to the R region of HIV-1. pCCL was derivedfrom plasmids 5′-GATATGATCAGATC-3′ (SEQ ID NO: 15) and 5′-CTGATCA-3′(SEQ ID NO: 16) and a three-part ligation along with a 0.54 kbAlwN-AvrII fragment and a 6.1 kb AvrII-BbsI fragment from pRRL

pRRL.SIN-45 was derived from pRRL by replacing the 493 bp BbsI-AlwNIfragment in the 3′ LTR with an oligonucleotide linker consisting ofsynthetic oligonucleotides, 5′-GATATGATCAGAGCCCTCAGATC-3′ (SEQ ID NO:17) and 5′-CTGAGGGCTCTGATCA-3′ (SEQ ID NO: 18) in a three-part ligationalong with a 0.54 kb AlwNI-AvrII fragment and a 6.1 kb AvrII-BbsIfragment from pRRL

pRRL.SIN-78 was derived from pRRL by replacing the 493 bp BbsI-AlwNIfragment in the 3′ LTR with an oligonucleotide linker consisting of,5′-GATATGATCAGGAGGCGTGGCCTGGGCGGGACTGGGGAGTGGCGAGCCC TCAGATC-3′ (SEQ IDNO: 19) and oligonucleotide5′-CTGAGGGCTCGCCACTCCCCAGTCCCGCCCAGGCCACGCCTCCTGATCA-3′ (SEQ ID NO: 20)in a three-part ligation along with a 0.54 kb AlwNI-AvrII fragment and a6.1 kb AvrII-BbsI fragment from pRRI.

EXAMPLE 5 Construction of Stable Lentiviral Packaging Cell) 0-28 and ofStable Producers of Lentiviral Vector

The 293G cell line was used to generate stable lentiviral packagingcells. 293G cells express the tetR/VP16 transactivator from the MDcassette (CMV promoter and intervening sequences—exons 2 and 3, intron2—and poly(A) site from the human β globin gene) and the VSV envelopefrom a minimal CMV promoter linked to a tandem repeat of seventetracycline operator sites (tet0). The expression of VSV G thus isregulated by the level of tetracycline in the culture medium, beingsuppressed in the presence of the antibiotic (Gossen & Bujard, Proc.Natl. Acad. Sci. USA (1992) 89:5547-5551); and Ory et al., supra (1997).The 293G cells were maintained routinely in DMEM/low glucose culturemedium supplemented with 10% donor calf serum and containing 1 μg/mltetracycline. A 15 cm plate of 293G cells were transfected usinglipofectamine (GIBCO BRL) with 13.36 μg of the packaging plasmidpCMVΔR8.74 and 1.33 μg of the selection plasmid pZeoSV2. The medium waschanged at 24 hr, and at 48 hr the cells were split into mediumcontaining 250 μg/ml zeocin and 1 μg/ml tetracycline. After 3-4 weeks inselection, 250 clones were picked and transferred to 96 well plates andthe medium screened for HIV-1 p24 Gag antigen by immunocapture using acommercially available kit. Fifty two p24 positive clones were grown upfor further analysis. The best 5 clones were determined to have p24values of 12-23 ng/ml. Of the 5 clones, 4 were positive for VSV.Gexpression after tetracycline withdrawal by Western blot analysis.

The four p24[VSV.G positive clones were analyzed further for the abilityto package lentiviral transfer vectors. The clones were infected withtransiently produced lentiviral vector (VSV.G pseudotype) containing anexpression cassette for the Green Fluorescent Protein of A. victoria(GFP) driven by the CMV promoter, at a multiplicity of infection of 10and in the presence of polybrene (8 μg/ml). The infected clones thenwere expanded and the tetracycline removed. After 72 hours of induction,a 24 hr medium collection was performed and the supernatants werefiltered and flash frozen. The frozen supernatants were titered on naiveHeLa cells for transduction of the GFP gene. By FACS analysis it wasdetermined that the population of cells (designated 10-28) created fromthe infection of packaging clone 10-28 had the highest titer of 5×104Transducing Units (T.U.)/ml.

The infected packaging population, 10-28, was used for the creation ofhigh titer producer clones of GFP lentiviral vector. 10-28 cells weresorted by FACS and the highest GFP expressing cells were retained andexpanded. That population then was infected serially (“pinged”) anadditional 4 times with transiently produced GFP lentiviral (VSV.Gpseudotype). After each infection the supernatants were collected aftera 72-96 hr of VSV.G induction. Supernatants were titered on HeLa cellsand analyzed for p24 content by immunocapture assay. Infectious titerspeaked after the third ping reaching 1.5×10⁶ T.U./ml (see FIG. 3). Thepopulation of cells from the third ping then were subcloned to isolatehigh titer vector producers.

EXAMPLE 6 Lentiviral Packaging Constructs

pMDLg/p is a CMV driven expression plasmid that contains only thegag/pol coding sequences from HIV-1. First, pkat2Lg/p was constructed byligating a 4.2 kb ClaI-EcoRI fragment from pCMVΔR8.74 with a 3.3 kbEcoRI-HindIII fragment from pkat2 (Finer et al., Blood (1994) 83: 43-50)and a 0.9 kb HindIII-NcoI fragment from pkat2 along with a NcoI-ClaI DNAlinker consisting of synthetic oligonucleotides5′-CATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGAT-3′ (SEQ ID NO: 21) and5′-CGATCTAATTCTCCCCCGCTTAATACTGACGCTCTCGCACC-3′ (SEQ ID NO: 22). Next,pMDLg/p was constructed by inserting the 4.25 kb EcoRI fragment frompkat2Lg/p into the EcoRI site of pMD-2. pMD-2 is a derivative of pMD.G(Ory et al., supra) in which the pXF3 plasmid backbone of pMD.G has beenreplaced with a minimal pUC18 (Invitrogen) plasmid backbone and the 1.6kb VSV.G encoding EcoRI fragment has been removed.

pMDLg/pRRE differs from pMDLg/p by the addition of a 374 bpRRE-containing sequence from HIV-1 (HXB2) immediately downstream of thepol coding sequences. To generate pMDLg/pRRE, the 374 bp NotI-HindIIIRRE-containing fragment from pHR3 was ligated into the 9.3 kb NotI-BglIIfragment of pVL1393 (Invitrogen) along with a HindIII-BglII DNA linkerconsisting of synthetic oligonucleotides 5′-AGCTTCCGCGGA-3′ (SEQ ID NO:23) and 5′-GATCTCCGCGGA-3′ (SEQ ID NO: 24) to generate pVL1393RRE (pHR3was derived from pHR2 by the removal of HIV env coding sequencesupstream of the RRE sequences in pHR2). A Not I site remains at thejunction between the gag and RRE sequences. pMDLg/pRRE then wasconstructed by ligating the 380 bp Eco RI-SstII fragment from pV1393RREwith the 3.15 kb SstII-NdeI fragment from pMD-2FIX (pMD-2FIX is a humanfactor IX containing a variant of pMD-2 which has an SstII site at the3′ end of the Factor IX insert), the 2.25 kb NdeI-AvrII fragment frompMDLg/p and the 3.09 kb AvrII-EcoRI fragment from pkat2Lg/p (Finer etal., supra).

pMDLg/pRRE. 2 is a gag/pol expressing lentiviral packaging vector inwhich the codons for the gag amino acids 2-13 have been mutated (withoutchanging the amino acids sequence). pMDLg/pRRE. 2 was generated byligating an 8.4 kb ClaI-Bsu36I fragment and a 1.4 kb Bsu36]-EcoRIfragment from pMDLg/pRRE with a DNA linker consisting of syntheticoligonucleotide,5′-aattcgagatctgccgccgccatgggagcccgggccagcgtcctgtctggaggggagctggac-3′(SEQ ID NO: 25) and5′-cggtccagctcccctccagacaggacgctggcccgggctcccatggcggcggcagatctcg-3′ (SEQID NO:26).

pMDLg/pRRE.3 is a gag/pol expressing lentiviral packaging vector inwhich the codons for the gag amino acids 2-7 have been mutated (withoutchanging the amino acids sequence) and in which gag coding sequences foramino acids from 8 to 87 of Gag polyprotein have been deleted.Previously described experiments which were conducted to study HIV-1 MAprotein functions (Reil et al., EMBO J. (1998) 17: 2699-708)demonstrated that deletion of amino acids from 8 to 87 of matrix protein(MA), which is part of Gag polyprotein, has no effect on efficiency ofwild type HIV-1 entry into infected cell, when analyzed virions werepseudotyped with VSV/G. pMDLg/pRRE. 3 was generated by ligating an 6.8kb SphI-Bsu36I fragment and a 1.4 kb Bsu36′-EcoRI fragment frompMDLg/pRRE with a 0.4 kb XbaI-SphI fragment from plasmid HXB 10ACT.A8-87 described in (Reil et al., supra) and a DNA linker consisting ofsynthetic oligonucleotides 5′aattcgagatctgccgccgccatgggagcccgggccagcgtc-3′ (SEQ ID NO: 27) and5′-ctagagacgctggcccgggctcccatggcggcggcagatctcg-3′ (SEQ ID NO: 28).

ptetMDrev is an expression vector in which HIV-1 Rev protein expressionis under the control of the tet inducible tet^(o)7/CMV hybrid promoter.The only HIV sequences contained in the vector are HXB2 rev cDNAcomprising the first (nucleotides 5937 through 6045) and second(nucleotides 8379 trough 8653) exons (Genbank accession number K03455).To generate ptetMDrev, the CMV enhancer/promoter of pMD-2 was replacedwith the tet^(o)/CMV hybrid promoter from ptet/splice (Gibco/BRL),yielding ptetMD. Next, ptetMDNcoI (ATG) was generated by inserting a DNAlinker consisting of synthetic oligonucleotides5′-aattcacgcgtgccgccaccatggcaggaagaagcggagacagcgacgaagacctcctcgcggccgccagtagctgt-3′(SEQ ID NO: 29) and5′-aattacagctactggcggccgcgaggaggtcttcgtcgctgtctccgcttcttcctgccatggtggcggcacgcgtg-3′(SEQ ID NO: 30) into EcoRI-digested ptetMD. Finally, ptetMDrev wasgenerated by ligating a 4.6 kb AlwNI-BamHI fragment and a 615 bp BamHI-BbsI fragment from ptetMDNcoI (ATG) with a 354 bp BbsI-AlWNI fragmentfrom pRSVrev (plasmid described in Dull et al., J. Virol. 1998) 72:8463-71).

EXAMPLE 7 Construction of Lentiviral Transfer Vectors

pHR7 is a maximally deleted lentiviral vector in which all HIV sequencesbetween nt 43 of the gag coding sequence and the transgene have beendeleted to further decrease homology between the transfer and packagingvectors. pHR7 was derived from pHR6 by ligating a 8.2 kb SacII-Not Ifragment and a 1.3 kb XhoI-SacII fragment from pHR6 with a DNA linkerconsisting of synthetic oligonucleotides 5′-GGCCATTGAC-3′ (SEQ ID NO:31) and 5′-TCGAGTCAAT-3′ (SEQ ID NO: 32).

pCCL7sinCMVGFPpre is a lentiviral vector which incorporates themaximally deleted 5′ untranslated region of pHR7 with a selfinactivating 3′ LTR, a CMV 5′ U3 and a post transcriptional regulatory(pre) element from the woodchuck hepatitis virus. To generatepCCL7sinCMVGFPpre, first a 329 bp AflII-XhoI fragment from pHR7 wasligated to a 1.9 kb XhoI-AvrII fragment and a 3.2 kb AvrII-AflII frompRRLsinl8hPGK.GFP to generate pRRL7sinhPGK.GFP. Next, the hPGK internalpromoter was replaced by a hCMV internal promoter by ligating a 606 bpClaI-BamHI fragment (in which the ClaI site was “filled”) frompRRLsinCMV.GFP with a 4.9 kb BamHI-AvaI fragment (in which the AvaI sitewas “filled”) from pRRL7sinhPGK.GFP to generate pRRL7sinhCMV.GFP. Next a600 bp SalI to EcoRI woodchuck hepatitis virus pre fragment (generatedby PCR using pWHV8 (Genbank assession #J04514) as the template withprimers 5′-tctagaggatccgtcgacaatcaacctctggattacaa-3′ (SEQ ID NO: 33) and5′gagctcgaattccaggcggggaggcggcccaa-3′ (SEQ ID NO: 34) followed bydigestion with SalI and EcoRI) was inserted into SalI and EcoRI digestedpRRL7sinhCMV.GFP to generate pRRL7sinhCMV.GFPpre. Next the 704 bp AflIIIto AflII fragment of pRRL7sinhCMV.GFP was replaced with the 1147 bpAflIII to AflII fragment from pCCL to generate pCCL7sinhCMV.GFPpre.

EXAMPLE 8 Construction of Conditional Self-Inactivating Vectors (cSIN)

pRRLsin36PGKGFPtet^(o)3′ is a lentiviral vector in which the 3′ LTRcontains a hybrid tet^(o)/HIV U3. The hybrid 3′ U3 consists of sevencopies of the tet operator (teto7) linked to the 36 nucleotides of the3′ portion of the HIV U3, which includes the “TATA” box.pRRLsin36PGKGFPtet^(o)3′ is a conditional self-inactivating (cSIN)vector that after transduction, can be activated to express full-lengthpackagable vector transcripts only in the presence of tetracyclineresponsive transactivator (tTA)—for example, after transduction of anappropriate packaging cell line expressing tTA. After transduction ofany cells not expressing tTA, the resulting 5′ tet^(o)/HIV U3 istranscriptionally non-functional, even in the presence of HIV Tatprotein, which is known to upregulate basal transcriptional activity ofheterologous promoters. That significantly reduces the chance ofmobilization of the vector genome even if transduced cells are infectedby the wild type HIV-1.

pRRLsin36PGKGFPtet^(o)3′ allows for a novel approach for a SIN vectordesign and vector system in general. The approach is based on the fact,that such a vector can be used for serial transductions (“pings”) intotTA-expressing packaging cell lines to obtain a high-titer producerclone while maintaining the SIN phenotype in non-tTA expressing targetcells.

To generate pRRLsin36PGKGFPtet 3′, first a 5.6 kb Asp718-BamHI fragmentfrom pRRL5sinl 8PGKGFP was ligated to a 303 bp XhoI-Asp718 fragment fromptet/splice (Gibco/BRL) along with the DNA linker consisting ofsynthetic oligonucleotides 5′-GATCCCGGGC-3′ (SEQ ID NO: 35) and5′-TCGAGCCCGG-3′ (SEQ ID NO: 36) to generate ptetINT (pRRL5sinl8PGKGFPis a vector in which the untranslated region of pRRLsinl8PGKGFP(Zufferey et. al., J. Virol., (1998) 72: 9873-9880) has been replacedwith the corresponding region from pHR5) Next a 2.8 kb AflIII-Asp718fragment from ptetINT was ligated to a 3.1 kb BclI-AflII fragment frompRRLsin36PGKGFP (Zufferey et. al. 1998) supra) along with the DNA linkerconsisting of synthetic oligonucleotides 5′-GTACCCGGGTCGAGTAGGCTT-3′(SEQ ID NO: 37) and 5′-GATCAAGCCTACTCGACCCGG-3′ (SEQ ID NO: 38) togenerate ptet36INT. Finally a 3.4 kb BamHI-AflIII fragment fromptet361NT was ligated to a 3.6 kb AflII-BclI fragment frompRRLsin36PGKGFP to yield pRRLsin36PGKGFPtet^(o)3′.

pCCL7sinCMVGFPpreTet^(o)3′ is a lentiviral transfer vector maximallydeleted in the 5′ untranslated region, in which the 3′ LTR ofpCCL7sinCMVGFPpre has been replaced with the tet-responsive 3′ LTR frompRRLsin36PGKGFPtet^(o)3′. pCCL7sinCMVGFPpreTet 3′ was generated byligating a 3.44 kb AflIII-EcoRI fragment from pCCL7sinCMVGFPpre with a3.5 kb EcoRI-AflIII fragment from pRRLsin36PGKGFPtet^(o)3′.

EXAMPLE 9

To isolate viral RNA, 0.45 micron-pore-size (Millipore) filteredsupernatants containing vector particles were adjusted for p24 contentand microcentrifuged at 14,000 rpm to pellet the virions. Supernatantswere aspirated and 50 μg of yeast RNA were added to each pellet ascarrier. Total RNA was isolated from the samples using RNAqueous kit(Ambion) according to manufacturer instructions. DNA probe template forin vitro transcription was prepared by two cycles of PCR using aLig′nScribe™ kit (Ambion) as instructed by the manufacturer. Probe 1 wasgenerated by PCR using primers 5′CATCAGGCCATATCACCTAGA-3′ (SEQ ID NO:39) and 5′-GTACTAGTAGTTCCTGCTATGT-3′ (SEQ ID NO: 40) and plasmidpCMVΔR8.74 to amplify a 298 bp fragment containing nucleotides 1215through 1513 of HIV-1 HXB2 (Genbank accession number K03455). Probe 2was generated by PCR using primers 5′-CTGCTGACATCGAGCTTGCTACA-3′ (SEQ IDNO: 41) and 5′-CTAGCTCCCTGCTTGCCCATACT-3′ (SEQ ID NO: 42) and plasmidpHR2 as template to amplify a 577 bp fragment containing nucleotides 336through 913 of HIV-1 HXB2 (Genbank accession number K03455). ³²Pantisense riboprobe then was synthesized by T7 RNA polymerase in thepresence of UTP (800 Ci/ml, DuPont NEN™). Full length probes were gelpurified and stored in 0.5 M ammonium acetate, 1 mM EDTA, and 0.2% SDSelution buffer at −20° C. RNA protection assay was performed using aHybSpeed™ kit (Ambion) according to manufacturer instructions. rnaseA/T1 mix (0.5 U/20 Upper reaction, Ambion) digestion protected probefragments were separated on 4% polyacrylamide, TBE and 8 M urea gels.For fragment size determination, ³²P-labeled an RNA markers weresynthesized on RNA Century template set and electrophoresed in parallel.For band detection and intensity quantification, dried gels were exposedeither to photofilm or a phosphorimager plate (Molecular Dynamics).

EXAMPLE 10

Transfer Vector Constructs. pHR′CMV-LacZ and pHR′CMV-Luciferase havebeen described (Naldini et al., Science, supra). pHR2 is a lentiviraltransfer vector in which the polylinker and downstream nef sequences upto the KpnI site of pHR′ have been replaced with aClaI/SpeI/SnaBI/SmaI/BamHI/SacII/EcoRI polylinker. pHR2 was generated byreplacing the 3.7 kb ClaI-SacI fragment of pHR′CMVlacZ with a 607 bpClaI-SacI fragment generated by PCR using pHR′CMVlacZ as the templatewith oligonucleotide primers5′-CCATCGATGGACTAGTCCTACGTATCCCCGGGGACGGGATCCGCGGAATTCCGTTTAAGACCAATGAC-3′ (SEQ ID NO: 43) and 5′-TTATAATGTCAAGGCCTCTC-3′ (SEQID NO: 44), followed by digestion with ClaI and SacI.

pHR2PGK-NGFR, pHR2CMV-NGFR and pHR2MFG-NGFR are lentiviral transfervectors in which the truncated low affinity NGF receptor (Bordignon etal., Hum. Gene Therap. (1995) 6:813-819) transgenes under the control ofthe murine PGK, human CMV or Moloney Leukemia Virus promoter,respectively, have been inserted into the polylinker of pHR2. ThepHR2PGK-NGFR transgene encodes no intron sequences while thepHR2CMV-NGFR vector includes the intron from plasmid pMD (Ory et al.,supra) and the pHR2MFG-NGFR vector contains the MLV intron from MFG-S(Ory et al., supra).

pRRL, pRLL, pCCL and pCLL are lentiviral transfer vectors containingchimeric Rous Sarcoma Virus (RSV)/HIV or CMV/HIV 5′ LTR's and vectorbackbones in which the SV40 polyadenylation and (enhancerless) origin ofreplication sequences have been included downstream of the HIV 3′ LTRreplacing most of the human sequence remaining from the HIV integrationsite. In pRRL, the enhancer and promoter (nucleotides −233 to −1relative to the transcriptional start site: Genbank accession numberJ02342) from the U3 region of RSV are joined to the R region of HIV-1LTR. In pRLL, the RSV enhancer (nucleotides −233 to −50) sequences arejoined to the promoter region (from position −78 bp relative to thetranscriptional start site) of HIV-1. In pCCL, the enhancer and promoter(nucleotides −673 to −1 relative to the transcriptional start site,Genbank accession number K03104) of CMV was joined to the R region ofHIV-1. In pCLL, the CMV enhancer (nucleotides −673 to −220) was joinedto the promoter region (position −78 bp) of HIV-1.

pHR2hPGK-GFP, pCCLhPGK-GFP, pCLLhPGK-GFP, pRRLhPGK-GFP, pRLLbPGK.GFP arelentiviral transfer vectors containing the enhanced Green FluorescentProtein (750 bp BamHI-NotI fragment from pEGFP-1 (Clontech)) codingregion under the control of the human PGK promoter (nucleotides 5-516,Genbank accession number M11958), inserted into the polylinker region ofeach parental vector. pRRLGFP was obtained by deletion of the XhoI-BamHIfragment containing the PGK promoter from pRRLhPGK-GFP.

pRRLhPGK.GFP.SIN-18 is a vector in which 3′ LTR sequences from 418 to−18 relative to the U3/R border have been deleted from pRLLhPGK.GFP.

Packaging Constructs. The tat-defective packaging construct pCMVΔR8.93was obtained by swapping a EcoRI-SacI fragment from the plasmidR7/pneo(−) (Feinberg et al., PNAS (1991) 88:4045-4049) with thecorresponding fragment of pCMVΔR8.91, a previously described Gag, Pol,Tat, and Rev expressing plasmid (Zufferey et al., 1997, supra). Thefragment has a deletion affecting the initiation codon of the tat geneand a frameshift created by the insertion of a MluI linker into theBsu36I site as described previously. pCMVΔR8.74 is a derivative ofpCMVΔR8.91 in which a 133 bp SacII fragment, containing a splice donorsite, has been deleted from the CMV-derived region upstream of the HIVsequences to optimize expression.

pMDLg/p is a CMV driven expression plasmid that contains only thegag/pol coding sequences from HIV-1. First, pkat2Lg/p was constructed byligating a 4.2 kb ClaI-Eco RI fragment from pCMVΔR8.74 with a 3. kbEcoRI-HindIII fragment from pkat2 (Finer et al., supra) and a 0.9 kbHindIII-NcoI fragment from pkat2 along with a NcoI-ClaI linkerconsisting of synthetic oligonucleotides,5′-CATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGAT-3′ (SEQ ID NO: 45) and5′-CGATCTAATTCTCCCCCGCTTAATACTGACGCTCTCGCACC-3′ (SEQ ID NO: 46). Next,pMDLg/p was constructed by inserting the 4.25 kb EcoRI fragment frompkat2Lg/p into the Eco RI site of pMD-2. pMD-2 is a derivative of pMD.G(Ory et al., supra) in which the pXF3 plasmid backbone of pMD.G has beenreplaced with a minimal pUC plasmid backbone and the 1.6 kb VSV.Gencoding EcoRI fragment has been removed.

pMDLg/pRRE differs from pMDLg/p by the addition of a 374 bpRRE-containing sequence from HIV-1 (HXB2) immediately downstream of thepol coding sequences. To generate pMDLg/pRRE, the 374 bp NotI-HindIIIRRE-containing fragment from pHR3 was ligated into the 9.3 kb NotI-BglIIfragment of pVL1393 (Invitrogen) along with a HindIII-BglIIoligonucleotide linker consisting of synthetic oligonucleotides5′-AGCTTCCGCGGA-3′ (SEQ ID NO: 47) and 5′ GATCTCCGCGGA-3′ (SEQ ID NO:48) to generate pVL1393RRE (pHR3 was derived from pHR2 by the removal ofHIV env coding sequences upstream of the RRE sequences in pHR2). A Not Isite remains at the junction between the gag and RRE sequences.pMDLg/pRRE was then constructed by ligating the 380 bp Eco RI-SstIIfragment from pV1393RRE with the 3.15 kb SstII-NdeI fragment frompMD-2FIX (pMD-2FIX is a human factor IX containing variant of pMD-2which has an SstII site at the 3′ end of the Factor IX insert), the 2.25kb NdeI-AvrII fragment from pMDLg/p and the 3.09 kb AvrII-EcoRI fragmentfrom pkatlLg/p (Finer et al., supra).

pRSV-Rev and pTK-Rev (generous gifts of T. Hope, Salk Institute) are revcDNA expressing plasmids in which the joined second and third exons ofHIV-1 rev are under the transcriptional control of either the RSV U3 orthe Herpes Simplex Virus 1 thymidine kinase promoter, respectively. Bothexpression plasmids utilize polyadenylation signal sequences from theHIV LTR in a pUC 118 plasmid backbone.

Vector production and assays. Vectors were produced by transienttransfection into 293T cells as previously described (Naldini et al.,PNAS, supra) with the following modifications. About 5×10⁶ 293T cellswere seeded in 10 cm dishes 24 hr prior to transfection in IMDM culturemedia (JRH Biosciences) with 10% FBS and penicillin (100 IU/ml) andstreptomycin (100 μg/ml) in a 5% CO₂ incubator and the culture mediumwas changed 2 hr prior to transfection. A total of 20 μg of plasmid DNAwas used for the transfection of one dish, 3.5 μg of the envelopeplasmid pMD.G, 6.5 μg of packaging plasmid and 10 μg of transfer vectorplasmid. The precipitate was formed by adding the plasmids to a finalvolume of 450 μl of 0.1×TE (TE: 10 mM Tris pH=8.0, 1 mM EDTA) and 50 μlof 2.5M CaCl₂, mixing well, then adding dropwise 500 μl of 2×HBS (281 mMNaCl, 100 mM HEPES, 1.5 mM Na₂HPO₄, pH=7.12) while vortexing, andimmediately adding the precipitate to the cultures. The medium (10 ml)was replaced after 14-16 hrs and the conditioned medium was collectedafter another 24 hr, cleared by low-speed centrifugation and filteredthrough 0.22 μm cellulose acetate filters. For in vitro experimentsserial dilutions of freshly harvested conditioned medium were used toinfect 10⁵ cells in a 6-well plate in the presence of 8 μg/ml polybrene.Viral p24 antigen concentration was determined by immunocapture(Alliance, DuPont-NEN). Vector batches were tested for the absence ofreplication-competent virus by monitoring p24 antigen expression in theculture medium of transduced SupT1 lymphocytes for three weeks. In allcases tested, p24 was undetectable (detection limit 3 pg/ml) once theinput antigen had been eliminated from the culture.

Northern Blot Analysis. Total RNA was isolated from 1-2×10⁷ cellsharvested at confluency using RNAsol B as suggested by the manufacturer.About 10-20 ug of RNA were loaded per well on 1% agarose gels usingNorthernMax (Ambion, Austin Tex.) reagents as described by themanufacturer. Transfer was to Zetabind membrane (Cuno Inc., MeridienConn.) either by capillary transfer or by pressure blotting(Stratagene). ³²P labelled probes were made by random priming.

Intracerebral injection of Vectors. Twelve Fischer 344 male ratsweighing approximately 220 g were obtained from Harlan Sprague-Dawley(Indianapolis, Ind.), housed with access to ad libitum food and water ona 12 hr light/dark cycle and were maintained and treated in accordancewith published NIH guidelines. All surgical procedures were performedwith the rats under isoflurane gas anesthesia using aseptic procedures.After a rat was anesthetized in a “sleep box” it was placed in a smallanimal stereotaxic device (Kopf Instruments, Tujunga, Calif.) using theearbars which do not break the tympanic membrane. The rats were randomlydivided into one control and four treatment groups. After the rats wereplaced in the stereotaxic frame, 2 μl of lentiviral vector concentratedby ultracentrifugation at 50,000×g for 140 min at 20° C. (Naldini etal., PNAS, supra) in phosphate buffered saline (PBS) were injectedconsecutively into the striatum in both hemispheres over 4 minutes at arate of 0.5 μl per minute (AP 0.0, LAT±3.0, DV−5.5, −4.5, −3.5 with theincisor bar set at −3.3 mm below the intraaural line; Paxinos & Watson,“The Rat Brain In Stereotaxic Coordinates” (1987) Academic Press, SD)using a continuous infusion system. During the injection, the needle wasslowly raised 1 mm in the dorsal direction every 40 seconds (3 mm totalwithdrawal). One minute after the cessation of the injection the needlewas retracted an additional 1 mm and then left in place for anadditional 4 minutes before being slowly withdrawn from the brain.

Histology. One month after vector injection, each animal was deeplyanesthetized with i.p. pentobarbital and perfused through the aorta withsterile PBS, followed by ice cold 4% paraformaldehyde (PFA) perfusion.The brains were removed from the skull, post-fixed in 4% PFA byimmersion for 24 hr and then transferred into a 30% sucrose/PBS solutionfor 3-4 days until the brains sank to the bottom of the containers. Thebrains then were frozen on dry ice and 40 μm thick coronal sections werecut on a sliding microtome. Sections were collected in series inmicrotitre-well plates that contained a glycerin based anti-freezesolution and they were kept at −30° C. until further processing.Immunocytochemistry was performed following the general proceduredescribed previously (Sternberger et al., J. Histochem. Cytochem. (1970)18:315-333). After several PBS rinses and an incubation in 3% hydrogenperoxide, the sections were placed in a 3% normal goat serum (NGS). Thesections then were incubated in the primary anti-GFP antibody (1:1000,Clontech, Palo Alto, Calif.) in 1% NGS and 0.1% Triton X-100 overnightat room temperature. After rinsing, the sections were incubated in thebiotinylated rabbit-anti-goat secondary antibody (Vector, Burlingame,Calif.) for 3 hours. After rinsing, the sections were incubated withhorseradish peroxidase streptavidin and then reacted using the purplechromagen kit VIP (Vector), mounted, dried, dehydrated, andcoverslipped.

Tat is required to produce vector of efficient transducing activity. Toinvestigate the role of Tat in the production of transducing particles,expression from lentiviral vectors was first examined by Northernanalysis. The patterns of RNA's induced by transfer vectors in which thetransgene was driven by an internal PGK, CMV, or retroviral MFG promoterwere studied in both producer and target cells. In transfected 293Tcells, expression occurred mainly from the internal promoter. When apackaging construct expressing both Tat and Rev was cotransfected, adramatic enhancement of transcription from the LTR was observed, with anaccumulation of unspliced vector RNA. In cells transduced with thevectors, that is, in the absence of Tat and Rev, transcription from theLTR was suppressed almost completely, the residual transcripts underwentsplicing and the internal promoter was responsible for most of theexpression.

A packaging plasmid carrying two mutations in tat (pCMVΔR8.93) then wasconstructed. The first mutation is a deletion of the T in the ATGinitiation codon of the tat gene, the second an insertion of a Mlu Ilinker producing a translation stop codon after residue 46 of the Tatprotein. These changes confer a tat-defective phenotype to HIV-1(Feinberg et al., supra). After transfection of the control ortat-defective packaging constructs into 293T cells, comparable yields ofvector particles were recovered in the culture medium, as assayed by theGag p24 antigen (see Table 2). Such Tat-independence was expected fromthe replacement of the HIV LTR by the constitutive CMV promoter in thepackaging construct. However, the particles produced in the absence ofTat had a dramatically reduced transducing activity (Table 3): 5 to 15%of that of particles produced by the control Tat-positive packagingconstruct.

TABLE 2 GFP transduction into HeLa cells by lentiviral vectors made bytransfer constructs with wild-type or 5′ chimeric LTR and packagingconstructs with or without a functional tat gene tat Gene End-pointTransduction Transfer in Packaging Titer p24 Antigen EfficiencyConstruct Construct (T.U./ml) (ng/ml) (T.U./ng p24) pHR2 + 4.1 × 10⁶ 29713,805 pHR2 − 2.4 × 10⁵ 545 440 PRRL + 1.3 × 10⁷ 546 23,810 PRRL − 4.9 ×10⁶ 344 14,244 Vectors carrying a PGK-eGFP expression cassette wereproduced by the transfection of the indicated transfer and packagingplasmid plus the pMD.G plasmid into 293T cells. Serial dilutions oftransfectant conditioned medium were incubated with HeLa cells, and thecultures were scored after 6 days. For calculating end-point titersamples were selected from the linear portion of the vector doseresponse curve. The average of duplicate determination is shown for arepresentative experiment of five performed. T.U. is transducing units.

TABLE 3 Transducing activity of lentiviral vectors made with and withouta functional tat gene in the packaging construct. Transducing Activity(Transduction Unit/ng p24) Transfer Target With Tat In Without Tat InVector Cells Packaging Construct Packaging Construct pHR′CMV-LacZ 293T1,056 ± 54^(a)   152 ± 26^(a) pHR2PGK-eGFP HeLa 5,666^(b) 384^(b)pHR′CMV- HeLa 3,000 ± 152^(c) 152 ± 26^(c) Luciferase pHR′CMV- HeLa-tat3,777 ± 348^(c) 486 ± 59^(c) Luciferase pHR′Luciferase^(d) HeLa  46 ±1^(c)   0.3 ± 0.003^(c) pHR′Luciferase^(d) HeLa-tat 3,296 ± 276^(c) 174± 75^(c) ^(a)LacZ transduction was measured by X-Gal staining and byexpressing the number of blue cell colonies as a function of the amountof p24 antigen in the inoculum ^(b)eGFP transduction was measured byFACS analysis, multiplying the fraction of fluorescent cells by thenumber of infected cells, and expressing the result as a function of theamount of p24 antigen in the inoculum ^(c)Luciferase transduction wasmeasured by the luminescence in relative units above background (RLU) of50 μl of culture extract and dividing the number of RLU × 10⁻² by thenumber of ng of p24 antigen in the inoculum ^(d)without internalpromoter Vectors were produced by the transfection of the indicatedtransfer vector, a packaging construct either with (pCMVΔR8.91) orwithout (pCMVΔR8.93) a functional tat gene and the pMD.G plasmid into293T cells. Serial dilutions of transfectant conditioned medium wereincubated with the indicated cells, and the cultures were scored after 3days. For calculating transduction activity, samples were selected fromthe linear portion of the vector dose response curve. The mean ± errorof triplicate determinations are shown for a,c, d; and the mean ofduplicate determinations is shown for b.

The tat-defective phenotype was tested to determine whether thephenotype could be rescued by complementation in target cells (Table 3).HeLa-tat cells, a cell line expressing Tat from the HIV-1 LTR (Felker etal., J. Virol. (1990) 64:3734-3741), were transduced by vectors producedwith or without Tat. The expression of Tat in target cells did notcompensate for the loss in transduction efficiency of vector producedwithout Tat.

As expected from the Northern analysis, functional inactivation of thetat gene resulted in a lower abundance of vector RNA in producer cells.That was indicated by the decrease in luciferase activity in cellsproducing a luciferase vector without internal promoter. There,transgene expression directly reflects the abundance of transcriptsoriginating from the LTR. 293T cells producing luciferase vectorswithout Tat had only 5% the luciferase content of cells producing thesame vector with Tat (1.0±0.2×10⁹ RLU/dish without Tat; 20.2±0.7×10⁹RLU/dish with Tat). The ratio corresponded very closely to that observedin cells transduced by either type of vector in the course of the sameexperiment (see Table 3), suggesting that the abundance of vector RNA inproducer cells is a rate-limiting factor in the transduction bylentiviral vectors.

It could be concluded that Tat is required in producer cells to activatetranscription from the HIV LTR and to generate vector particles with ahigh transducing activity.

The tat requirement is offset by placing a constitutive promoterupstream of the transfer vector. If the only function of Tat istrans-activation of vector transcription from the LTR, the tat-defectivephenotype should be rescued by placing a strong constitutive promoterupstream of the vector transcript. Three transcriptional domains havebeen identified in the HIV promoter in the U3 region of the LTR: thecore or basal domain, the enhancer and the modulatory domain (Luciw,supra). Transcription starts at the U3/R boundary, the first nucleotideof R being numbered 1. The core promoter contains binding sites for theTATA-binding protein (−28 to −24) and SP-1 (three binding sites between−78 to 45). The enhancer contains two binding sites for NF-κB whichoverlap with a binding site for NFATc (−104 to −81). The modulatorydomain contain binding sites for several cellular factors, includingAP-1 (−350 to −293), NFAT-1 (−256 to −218), USF-1 (−166 to −161), Ets-1(−149 to −141) and LEF (−136 to −125). A panel of 5′ chimeric transferconstructs carrying substitutions of either all or part of the U3 regionof the 5′ LTR was generated. All substitutions were made to preserve thetranscription initiation site of HIV. Partial substitutions joined newenhancer sequences to the core promoter of the HIV LTR (−78 to 1), whilefull substitutions replaced also the promoter. RLL and RRL vectorscarried the enhancer or the enhancer and promoter, respectively, of Roussarcoma virus (RSV); and CLL and CCL vectors carried the enhancer or theenhancer and promoter of human CMV.

Control pHR2 and 5′ chimeric transfer constructs carrying a PGK-eGFPexpression cassette were tested by transfection of 293T cells withcontrol or tat-defective packaging constructs and the expression of theeGFP transgene was analyzed by FACS. The RRL chimeric construct yieldedhigher eGFP expression level than the pHR2 vector, reflecting theconstitutive transcriptional activity of the new sequence.Interestingly, the chimeric vector also displayed upregulation by Tat,as shown by the increased eGFP expression of cells cotransfected withthe control packaging construct. Tat upregulation was proven to be adirect effect by transfecting a pRRL-eGFP vector lacking an internalpromoter with control or tat-defective packaging constructs andanalyzing GFP expression by FACS. Comparable results were obtained withthe other chimeric LTR vectors. Vector particles then were collectedfrom the transfected producer cells and assayed for transduction of eGFPinto HeLa cells and human primary lymphocytes (PBL). As shown in Table4, all vectors had efficient transducing activity, as assessed byend-point titration on HeLa cells, or maximal transduction frequency ofPBL. The vector produced by the pRRL chimera was as efficient as thatproduced by the pHR2 construct and was selected to test transductionindependent of Tat. As shown in Table 2, the pRRL construct yieldedvector of only slightly reduced transducing activity (60%) when thepackaging construct was tat-defective. The residual effect of Tat ontransduction was in agreement with the ability of Tat to upregulatetranscription from the chimeric LTR. Tat upregulation was proven to be adirect effect by transfecting a pRRL-eGFP vector lacking an internalpromoter with control or tat-defective packaging constructs andanalyzing GFP expression by FACS.

TABLE 4 GFP transduction by lentiviral vectors made by transferconstructs with wild-type or a 5′ chimeric LTR End-Point Titer onTransduction Efficiency on Transfer HeLa cells Human LymphocytesConstruct (T.U./ml)^(a) (% positive cells)^(b) pHR2 2.3 × 10⁷ 30% PCCL4.6 × 10⁶ 14% PCLL 7.9 × 10⁶ 18% PRRL 1.8 × 10⁷ 29% PRLL 8.9 × 10⁶ 18%^(d)end-point titer was determined by multiplying the percent offluorescent cells for the vector dilution and the number of infectedcells. Samples were selected from the linear portion of the vectordose-response curve ^(b)percentage of fluorescent human peripheral bloodlymphocytes after infection of 10⁶ cells with 1 ml of vector containingmedium. Primary human T lymphocytes were isolated and transduced aspreviously described (Finer et al., supra) Vectors carrying a PGK-eGFPexpression cassette were produced by the transfection of the indicatedtransfer construct, the packaging plasmid pCMVΔR8.91 and the envelopeplasmid pMD.G into 293T cells. Fluorescent cells were scored by FACSanalysis 6 days after transduction. The average of duplicatedetermination is shown for a representative experiment of threeperformed.

The use of the chimeric LTR construct allowed removal of Tat from thepackaging system with a minimal loss in the transduction efficiency ofthe vector in vitro. To test vector performance in the more challengingsetting of in vivo delivery into brain neurons, high-titer vector stockswere generated from the pHR2 and pRRL construct with and without Tat.The four stocks of eGFP vector were matched for particle content by p24antigen and injected bilaterally in the neostriatum of groups of threeadults rats. The animals were sacrificed after 1 month and serialsections of the brain were analyzed for eGFP fluorescence andimmunostained by antibodies against eGFP. The results obtained in vivomatched the in vitro data. Vector produced by the pHR2 construct onlyachieved significant transduction of the neurons when packaged in thepresence of Tat. Vector produced by the pRRL chimera was as wellefficient when made with or without Tat. The transduction extendedthroughout most of the striatum and reached a very high density ofpositive cells in the sections closest to the injection site. No signsof pathology were detectable in the injected tissue, except for a smalllinear scar marking the needle track, by hematoxylin and eosin stainingof the sections.

The results provide evidence that Tat is dispensable for efficienttransduction by a lentiviral vector.

A split-genome conditional packaging system. The possibility of deletingthe tat gene prompted design of another packaging component of the HIVvector system in which two separate non-overlapping expression plasmids,one for the gag-pol gene and the other for the rev gene, were used. Thegag-pol reading frames were expressed within the context of the MDcassette, which employs the CMV promoter and intervening sequence andthe human β globin poly(A) site (Ory et al., supra). All the HIVsequences upstream of the gag initiation codon were removed and theleader was modified for optimal fit to the Kozak consensus fortranslation. The construct, however, expressed almost no p24 antigenwhen transfected alone in 293T cells. That observation is in agreementwith the previously reported presence of cis-repressive or inhibitorysequences in the gag/pol gene (Schneider et al., J. Virol. (1997) 71:4892-4903; and Schwartz et al., J. Virol. (1992) 66:7176-7182). The HIVRRE element was then inserted downstream of the pol gene and theresulting plasmid was cotransfected with a rev expression vector (Table5).

High levels of p24 antigen production were observed, the highest yieldsbeing obtained when rev was driven by an RSV promoter. When the gag/poland the rev constructs were cotransfected with the RRL chimeric transfervector and the VSV G-expressing plasmid, high titer vector was obtainedin the culture medium. Both the yield of particles and their transducingefficiency were similar to those obtained with previous versions of thesystem. Northern analysis of producer cells confirmed that unsplicedvector genomic RNA accumulated only in the presence of Rev. Thus, boththe expression of the gag-pol genes and the accumulation of packageablevector transcripts are dependent on trans-complementation by a separateRev expression construct. Such a conditional packaging system providesan important safety feature unavailable to oncoretroviral vectors.

TABLE 5 GFP transduction into HeLa cells by lentiviral vectors made bylinked or split packaging constructs and a pRRL transfer construct. p24End-point Transduction Packaging Separate rev Antigen Titer EfficiencyConstruct Plasmid^(a) (ng/ml) (T.U./ml) (T.U./ng p24) pCMVΔR8.74 — 3641.07 × 10⁷ 29,436 pMDLg/pRRE — <0.1 N.D.^(b) N.A. pMDLg/pRRE TK-Rev 5 μg29  6.9 × 10⁵ 23,793 pMDLg/pRRE TK-Rev 12 μg 94 2.02 × 10⁶ 21,489pMDLg/pRRE RSV-Rev 2.5 μg 774  1.0 × 10⁷ 13,495 pMDLg/pRRE RSV-Rev 5 μg776  7.6 × 10⁶  9,761 pMDLg/pRRE RSV-Rev 12 μg 565  4.8 × 10⁶  8,495^(a)the promoter driving the expression of a synthetic rev cDNA and theamount of plasmid transfected are indicated Vectors carrying a PGK-eGFPexpression cassette were produced by the transfection of aself-inactivating pRRL transfer construct (with a deletion in the 3′ LTR53), the indicated packaging and rev plasmids and the pMD.G plasmid into293T cells. Serial dilutions of transfectant conditioned medium wereincubated with HeLa cells and the cultures were scored after 6 days. Forcalculating end-point titer samples were selected from the linearportion of the vector dose response curve. The average of duplicatedetermination is shown for a representative experiment of threeperformed. ^(b)N.D.: none detected. The detection limit of the assay was10² T.U./ml.

EXAMPLE 11

In another embodiment of the invention, pRRLsin36PGKGFPtet^(o)3′ is alentivirus vector in which the 3 LTR contains a hybrid tet^(o)/HIV U3.The 3′ U3 consists of seven copies of the tet operator (teto7) linked tothe 3′ 36 nucleotides of the HIV U3 including the “tata” box.pRRLsin36PGKGFPtet^(o)3′ is a conditional self inactivating (SIN) vectorthat, after transduction, can be activated to express full-lengthpackagable vector transcripts only in the presence of tet-transactivator(tta)—for example, after transduction of an appropriate tta expressingpackaging cell line. After transduction of any cell not expressing tta,the resulting 5′ teto7/HIV U3 is essentially non-functional, even in thepresence of HIV tat, significantly reducing the chance of mobilizationof the vector genome. pRRLsin36PGKGFPtet^(o)3′ allows for a SIN vectorwhich can be serially transduced (“pinged”) into a tta-expressingpackaging cell line to obtain a high-titer producer clone whilemaintaining the SIN phenotype in non-tta expressing target cells. Togenerate pRRLsin36PGKGFPtet^(o)3′, first a 5.6 kb Asp718-BamHI fragmentfrom pRRL5sinl8PGKGFP was ligated to a 303 bp XhoI-Asp718 fragment fromptetsplice along with a DNA linker consisting of syntheticoligonucleotides 5′GATCCCGGGC-3′ (SEQ ID NO: 49) and 5′TCGAGCCCGG-3′(SEQ ID NO: 50) to generate ptetINT (pRRL5sinl8PGKGFP is a vector inwhich the untranslated region of pRRLsinl8PGKGFP (Zufferey et al., J.Virol (1998) 72: 9873-9880) has been replaced with the correspondingregion from pHR5) Next a 2.8 bk AflII-Asp718 fragment from ptetINT wasligated to a 3.1 kb BclI-AflII fragment from pRRLsin36PGKGFP (Zuffereyet al. (1998) supra) along with a DNA linker consisting of syntheticoligonucleotide 5′-GTACCCGGGTCGAGTAGGCTT-3′ (SEQ ID NO: 51) and5′-GATCAAGCCTACTCGACCCGG-3′ (SEQ ID NO: 52) to generate ptet361NT.Finally, a 3.4 kb BamHI-AflII fragment from ptet361NT was ligated to a3.6 kb AflII-BclI fragment from pRRLsin36PGKGFP to yieldpRRLsin36PGKGFPtet^(o)3′.

Another such vector is maximally deleted in the 5′ untranslated region.The 3′ LTR of pCCL7sinCMVGFPpre has been replaced with thetet-responsive 3′ LTR from pRRLsin36PGKGFPtet^(o)3′.pCCL7sinCMVGFPpreTet^(o)03′ was generated by ligating a 3.44 kbAflIII-EcoRI fragment from pCCL7sinCMVGFPpre with a 3.5 kb EcoRI-AflIIIfragment from pRRLsin36PGKGFPtet^(o)3′.

EXAMPLE 12

To generate vector stocks containing a tetracycline inducible promotersequence in the U3 region of the mRNA, the following plasmids weretransfected: 10 μg of pRRLsin36PGKGFP, pRRLhPGKGFP orpRRLsin36PGKGFPtet; 6.5 μg of pMDLg/pRRE; and 3 μg of pMD.G into 293Tcells. Vector stocks containing mRNA derived from the pRRLhPGKGFPconstruct served as a positive (non-regulatable) control. Vector stockscontaining mRNA derived from the pRRLsin36PGKGFP construct served as anegative control (since on tranduction, copying by reverse transcriptase(RT) of a deleted U3 region to the 5′ region of the integrated vectorDNA would render the resulting LTR transcriptionally non-functional).

Supernatants of the transfected cells were collected, 0.22 micron poresize filtered and used for rounds of pinging of a 2^(nd) generationpackaging cell line at MOI=5 TU/cell (multiplicity of infection) eachping. Cells were cultured for an additional 2 weeks, split into 10 cmdishes at 50 to 70% confluence and induced for vector production byremoving tetracycline from the medium. Supernatants of the inducedpromoter cells were collected as indicated in FIG. 10 and assayed forp24 and titer. Titer determination was done by infection of indicatorHeLa cells with limited dilutions of assayed vector preparations.Percentage of transduced cells were scored by FACS.

As can be seen from FIG. 10, vector production and titers of vectorparticles for tetracycline regulatable transfer vectors were comparableto those of the positive control.

In contrast to an HIV-1 derived LTR, transcriptional activity of the LTRof such a vector in the cells lacking tTA was not detected by Northernanalysis of transduced cells (FIG. 11), or when GFP expression levelswere analyzed by FACS (FIG. 12) even on infection of transduced cells bythe wild type HIV-1. Total RNA was extracted (by standard techniques)from transduced cells and assayed with ³²P-labeled DNA probe. Probe wasgenerated by a random priming kit (HighPrime™, Boeringer Mannheim) usinga BamHI-NotI fragment of the GFP coding sequence of plasmidpRRLsinPGKGFP was the template.

As can be seen in FIG. 11, in contrast to a vector with an HIV-1 LTR, noLTR driven mRNA could be detected for both the control and tetracyclineresponsive vectors. Consistent with those results, FACS analysis (FIG.12) also showed that GFP expression was upregulated by HIV-1 infectiononly in cells transduced by the vector with a full length HIV-1 LTR.Thus, such regulatable vectors retain the SIN phenotype.

All publications and patents cited in the instant specification areherein incorporated by reference in their entirety as if each individualpublication or patent application were specifically and individuallyindicated to be incorporated by reference.

As will be apparent to those skilled in the art to which the inventionpertains, the instant invention may be embodied in forms other thanthose specifically disclosed above, for example to transfect andtransduce other mammalian cell types, without departing from the spiritor essential characteristics of the invention. The particularembodiments of the invention described above, are, therefore, to beconsidered as illustrative and not restrictive. The scope of the instantinvention is as set forth in the appended claims rather than beinglimited to the examples contained in the foregoing description.

1. A lentiviral vector system comprising a lentiviral packaging systemand a lentiviral transfer vector comprising a heterologous gene operablylinked to a regulatory element, wherein the lentiviral packaging systemcomprises a structural lentiviral vector system comprising a firstlentiviral vector that encodes a structural gene selected from a gaggene, a pol gene or both gag and pol genes; and a regulatory lentiviralvector comprising a rev gene, wherein the regulatory lentiviral vectoris provided on a separate construct from the structural lentiviralvector system, wherein the lentiviral transfer vector comprises a 5′ LTRand a 3′ LTR, wherein the regulatory element is a heterologousregulatory element operable in a mammalian cell, wherein a part or allof a regulatory element of the U3 region of the 5′ LTR is replaced bythe heterologous regulatory element, wherein a part or all of the U3region of the 3′ LTR is replaced by a heterologous inducible regulatoryelement that is activated only in the presence of an activator expressedin trans, and wherein the lentiviral vector system lacks a functionaltat gene.
 2. The lentiviral vector system of claim 1, wherein theheterologous inducible regulatory element comprises a tet operator.
 3. Amethod of producing a recombinant lentivirus comprising: (a)transfecting a packaging host cell with the lentiviral vector system ofclaim 1; and (b) recovering the recombinant lentivirus produced by thetransfected packaging host cell.
 4. The lentiviral vector system ofclaim 1, wherein the regulatory lentiviral vector further comprises aheterologous regulatory element operably linked to the rev gene.
 5. Thelentiviral vector system of claim 1, wherein the structural lentiviralvector system further comprises a regulatory response element (RRE)downstream of the structural gene.
 6. The lentiviral vector system ofclaim 1, wherein the structural lentiviral vector system furthercomprises a heterologous regulatory element operably linked to thestructural gene.
 7. The lentiviral vector system of claim 1, wherein thetat gene is deleted.
 8. The lentiviral vector system of claim 1, whereinthe tat gene is mutated.
 9. The lentiviral vector system of claim 1,which lacks a functional HIV env gene.
 10. The lentiviral vector systemof claim 1, further comprising a viral env gene that is derived from adifferent virus than the structural genes.
 11. The lentiviral vectorsystem of claim 1, which lacks functional vif, vpr, vpu and nef genes.12. The lentiviral vector system of claim 1, wherein the lentivirus ishuman immunodeficiency virus (HIV).
 13. The lentiviral vector system ofclaim 2, wherein the heterologous inducible regulatory element comprisesseven copies of a tet operator (tet^(o)7).
 14. The lentiviral vectorsystem of claim 13, wherein the tet^(o)7 is linked to a part of the 3′U3 region that comprises a TATA box sequence.
 15. The lentiviral vectorsystem of claim 4, wherein the heterologous regulatory element operablylinked to the rev gene comprises a RSV U3 or a herpes simplex virusthymidine kinase (HSVtk) promoter.
 16. The lentiviral vector system ofclaim 6, wherein the heterologous regulatory element operably linked tothe structural gene comprises a CMV promoter.
 17. The lentiviral vectorsystem of claim 10, wherein the env gene is provided on a vector otherthan the first lentiviral vector.
 18. The lentiviral vector system ofclaim 12, wherein the HIV is HIV-1.